Antibodies specific for CD70 and their uses

ABSTRACT

The present invention provides antibodies that specifically bind to CD70 (Cluster of Differentiation 70). The invention further provides bispecific antibodies that bind to CD70 and another antigen (e.g., CD3). The invention further relates to antibody encoding nucleic acids, and methods of obtaining such antibodies (monospecific and bispecific). The invention further relates to therapeutic methods for use of these antibodies for the treatment of CD70-mediated pathologies, including cancer.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent Appl. No.62/641,873, filed Mar. 12, 2018, and U.S. Provisional Patent Appl. No.62/625,019, filed Feb. 1, 2018, each of which is incorporated herein byreference in its entirety.

REFERENCE TO SEQUENCE LISTING

This application is being filed electronically via EFS-Web and includesan electronically submitted sequence listing in .txt format. The .txtfile contains a sequence listing entitled “ALGN-015_02 US_SL.txt”created on Jan. 30, 2019, and having a size of 223 kilobytes. Thesequence listing contained in this .txt file is part of thespecification and is incorporated herein by reference in its entirety.

FIELD

The present invention relates to antibodies, e.g., full lengthantibodies or antigen binding fragments thereof, that specifically bindto Cluster of Differentiation 70 (CD70). The invention further relatesto heteromultimeric antibodies (e.g., bispecific antibodies) comprisingCD70 antibody on one arm. Compositions comprising the CD70 antibodies,methods for producing and purifying such antibodies, and their use indiagnostics and therapeutics are also provided.

BACKGROUND

Renal Cell Carcinoma (RCC) is a cancer that originates in the renalcortex and accounts for about 90% of cancers in the kidney. Based onhistology, RCC can be classified into several sub-types, of which ClearCell Renal Cell Carcinoma (ccRCC) is the most common and leads to themost deaths. Each year, over 320,000 cases of RCC are reported worldwideleading to roughly 140,000 deaths. The incidence of RCC has risensteadily over the last 10 years and accounts for 2-3% of all adultmalignancies. Patients with early stage localized tumors can opt forsurgical resection; however, localized disease can undergo earlyhematogenous dissemination leading to metastasis. Sites of earlymetastases include lungs, lymph nodes, liver, bone, and brain; lesscommonly the adrenal glands, and the contralateral kidney. Patients withadvanced disease face high morbidity rates with a 5-year median survivalrate of 53% for stage III disease and only 8% for metastatic disease.Current first-line treatment options for advanced disease include smallmolecule Tyrosine Kinase Inhibitors (TKIs) such as sunitinib andpazopanib that target Vascular Endothelial Growth Factor (VEGF)receptor, monoclonal antibody targeting VEGF such as bevacizumab,mammalian target of Rapamycin (mTOR) inhibitor temsirolimus, as well ashigh dose IL-2. Although these VEGF-targeted therapies have improvedover-all survival, long-term drug resistance leads to disease relapseand treatment for advanced disease still remains an unmet need (see,e.g., Zarrabi, K. et al., Journal of Hematology and Oncology, 10:38(2017)).

Cluster of Differentiation 70 (CD70, CD27LG or TNFSF7) is a member ofthe tumor necrosis factor (TNF) superfamily and the ligand for CD27, aTNF superfamily receptor. The transient interaction between CD27 andCD70 provides T cell costimulation complementary to that provided byCD28. CD70 is expressed on hematological cancers such as Non-Hodgkin'sLymphoma and Hodgkin's disease as well as on solid tumors such asGlioblastoma and Renal Cell Carcinoma; with its expression on ccRCCbeing nearly uniform (see e.g., Grewal I., et al., Expert Opinion onTherapeutic Targets, 12(3): 341-351 (2008)).

CD70 bispecific antibody in the form of T-cell engaging bispecificapproach has been developed recently. However, a limitation of manybispecific formats is that they are of small molecular weight, and ofshort half-life, thus requiring continuous infusion. Accordingly, thereremains a need for antibodies (e.g., monospecific or bispecific)treating cancer where CD70 is expressed and in particular mRCC withimproved efficacy and safety profile, and suitable for use with humanpatients.

SUMMARY

The invention disclosed herein is directed to antibodies (e.g.,monospecific or bispecific antibodies) that specifically bind to Clusterof Differentiation 70 (CD70). In some embodiments, the CD70 antibodiesas described herein in the full-length bispecific format have longerhalf-life, minimized Fc-interaction, and minimized non-specific cytokinerelease in vivo via interaction with immune cells.

Accordingly, in one aspect, the invention provides an isolated antibodywhich specifically binds to CD70, wherein the antibody comprises (a) aheavy chain variable (VH) region comprising (i) a VH complementaritydetermining region one (CDR1) comprising the sequence shown in SEQ IDNO: 49, 50, 51, 55, 56, 57, 61, 62, 63, 67, 68, 69, 73, 74, 75, 79, 80,81, 85, 86, 87, 91, 92, 93, 97, 98, 99, 103, 104, 105, 109, 110, 111,115, 116, 117, 121, 122, 123, 127, 128, 129, 133, 134, 135, 139, 140,141, 145, 146, 147, 151, 152, 153, 157, 158, 159, 163, 164, 165, 169,170, 171, 175, 176, 177, 181, 182, 183, 187, 188, 189, 332, 333, 334,338, 339, 340, 344, 345, 346, 350, 351, 352, 356, 357, 358, 362, 363,364, 368, 369, 370, 374, 375, 376, 380, 381, 382, 386, 387, 388, 392,393, 394, 398, 399, 400, 404, 405, 406, 410, 411, 412, 416, 437, 418,422, 423, 424, 428, 429, 430, 434, 435, 436, 440, 441, 442, 446, 447,448, 452, 453, 454, 458, 459 or 460; (ii) a VH CDR2 comprising thesequence shown in SEQ ID NO: 52, 53, 58, 59, 64, 65, 70, 71, 76, 77, 82,83, 88, 89, 94, 95, 100, 101, 106, 107, 112, 113, 118, 119, 124, 125,130, 131, 136, 137, 142, 143, 148, 149, 154, 155, 160, 161, 166, 167,172, 173, 178, 179, 184, 185, 190, 191, 335, 336, 341, 342, 347, 348,353, 354, 359, 360, 365, 366, 371, 372, 377, 378, 383, 384, 389, 390,395, 396, 401, 402, 407, 408, 413, 414, 419, 420, 425, 426, 431, 432,437, 438, 443, 444, 449, 450, 455, 456, 461 or 462; and iii) a VH CDR3comprising the sequence shown in SEQ ID NO: 54, 60, 66, 72, 78, 84, 90,96, 102, 108, 114, 120, 126, 132, 138, 144, 150, 156, 162, 168, 174,180, 186, 192, 337, 343, 349, 355, 361, 367, 373, 379, 385, 391, 397,403, 409, 415, 421, 427, 433, 439, 445, 451, 457 or 463; or a lightchain variable (VL) region comprising (i) a VL CDR1 comprising thesequence shown in SEQ ID NO: 193, 196, 199, 202, 205, 208, 211, 214,217, 220, 223, 226, 229, 232, 235, 238, 241, 244, 247, 250, 253, 256,259, 262, 464, 467, 470, 473, 476, 479, 482, 485, 488, 491, 494, 497,500, 503, 506, 509, 512, 515, 518, 521, 524 or 527; (ii) a VL CDR2comprising the sequence shown in SEQ ID NO: 194, 197, 200, 203, 206,209, 212, 215, 218, 221, 224, 227, 230, 233, 236, 239, 242, 245, 248,251, 254, 257, 260, 263, 465, 468, 471, 474, 477, 480, 483, 486, 489,492, 495, 498, 501, 504, 507, 510, 513, 516, 519, 522, 525 or 528; and(iii) a VL CDR3 comprising the sequence shown in SEQ ID NO: 195, 198,201, 204, 207, 210, 213, 216, 219, 222, 225, 228, 231, 234, 237, 240,243, 246, 249, 252, 255, 258, 261, 264, 466, 469, 472, 475, 478, 481,484, 487, 490, 493, 496, 499, 502, 505, 508, 511, 514, 517, 520, 523,526 or 529.

In another aspect, provided is an isolated antibody which specificallybinds to CD70, wherein the antibody comprises: a VH region comprising aVH CDR1, VH CDR2, and VH CDR3 of the VH sequence shown in SEQ ID NO: 2,4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40,42, 44, 46, 48, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309,311, 313, 315, 317, 319, 321, 323, 325, 327, 329 or 331; or a VL regioncomprising VL CDR1, VL CDR2, and VL CDR3 of the VL sequence shown in SEQID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33,35, 37, 39, 41, 43, 45, 47, 288, 290, 292, 294, 296, 298, 300, 302, 304,306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328 or 330. Insome embodiments, the VH region as described herein comprises a variantwith one or several conservative amino acid substitutions in residuesthat are not within a CDR or the VL region as described herein comprisesa variant with one or several amino acid substitutions in amino acidsthat are not within a CDR. For example, in some embodiments, the VH orVL region can comprise an amino acid sequence described above or avariant thereof with no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1conservative substitutions in residues that are not within a CDR.

In some embodiments, provided is an isolated antibody which specificallybinds to CD70, wherein the antibody comprises: a VH region comprisingthe sequence shown in SEQ ID NO: 18; or a VL region comprising thesequence shown in SEQ ID NO: 17.

In some embodiments, provided is an antibody which specifically binds toCD70 and competes with an isolated antibody provided herein whichspecifically binds to CD70.

In another aspect, provided is a bispecific antibody wherein thebispecific antibody is a full-length antibody, comprising a firstantibody variable domain of the bispecific antibody specifically bindingto a target antigen (e.g., CD70), and comprising a second antibodyvariable domain of the bispecific antibody capable of recruiting theactivity of a human immune effector cell by specifically binding to aneffector antigen (e.g., Cluster of differentiation 3 (CD3)) located onthe human immune effector cell. In some embodiments, the first antibodyvariable domain comprises a heavy chain variable (VH) region comprisinga VH CDR1, VH CDR2, and VH CDR3 of the VH sequence shown in SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38,40, 42, 44, 46, 48, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307,309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329 or 331; or a lightchain variable (VL) region comprising VL CDR1, VL CDR2, and VL CDR3 ofthe VL sequence shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19,21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 288, 290, 292,294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320,322, 324, 326, 328 or 330. In some embodiments, the first antibodyvariable domain comprises (a) a heavy chain variable (VH) regioncomprising (i) a VH complementarity determining region one (CDR1)comprising the sequence shown in SEQ ID NO: 49, 50, 51, 55, 56, 57, 61,62, 63, 67, 68, 69, 73, 74, 75, 79, 80, 81, 85, 86, 87, 91, 92, 93, 97,98, 99, 103, 104, 105, 109, 110, 111, 115, 116, 117, 121, 122, 123, 127,128, 129, 133, 134, 135, 139, 140, 141, 145, 146, 147, 151, 152, 153,157, 158, 159, 163, 164, 165, 169, 170, 171, 175, 176, 177, 181, 182,183, 187, 188, 189, 332, 333, 334, 338, 339, 340, 344, 345, 346, 350,351, 352, 356, 357, 358, 362, 363, 364, 368, 369, 370, 374, 375, 376,380, 381, 382, 386, 387, 388, 392, 393, 394, 398, 399, 400, 404, 405,406, 410, 411, 412, 416, 437, 418, 422, 423, 424, 428, 429, 430, 434,435, 436, 440, 441, 442, 446, 447, 448, 452, 453, 454, 458, 459 or 460;(ii) a VH CDR2 comprising the sequence shown in SEQ ID NO: 52, 53, 58,59, 64, 65, 70, 71, 76, 77, 82, 83, 88, 89, 94, 95, 100, 101, 106, 107,112, 113, 118, 119, 124, 125, 130, 131, 136, 137, 142, 143, 148, 149,154, 155, 160, 161, 166, 167, 172, 173, 178, 179, 184, 185, 190, 191,335, 336, 341, 342, 347, 348, 353, 354, 359, 360, 365, 366, 371, 372,377, 378, 383, 384, 389, 390, 395, 396, 401, 402, 407, 408, 413, 414,419, 420, 425, 426, 431, 432, 437, 438, 443, 444, 449, 450, 455, 456,461 or 462; and iii) a VH CDR3 comprising the sequence shown in SEQ IDNO: 54, 60, 66, 72, 78, 84, 90, 96, 102, 108, 114, 120, 126, 132, 138,144, 150, 156, 162, 168, 174, 180, 186, 192, 337, 343, 349, 355, 361,367, 373, 379, 385, 391, 397, 403, 409, 415, 421, 427, 433, 439, 445,451, 457 or 463; or (b) a light chain variable (VL) region comprising(i) a VL CDR1 comprising the sequence shown in SEQ ID NO: 193, 196, 199,202, 205, 208, 211, 214, 217, 220, 223, 226, 229, 232, 235, 238, 241,244, 247, 250, 253, 256, 259, 262, 464, 467, 470, 473, 476, 479, 482,485, 488, 491, 494, 497, 500, 503, 506, 509, 512, 515, 518, 521, 524 or527; (ii) a VL CDR2 comprising the sequence shown in SEQ ID NO: 194,197, 200, 203, 206, 209, 212, 215, 218, 221, 224, 227, 230, 233, 236,239, 242, 245, 248, 251, 254, 257, 260, 263, 465, 468, 471, 474, 477,480, 483, 486, 489, 492, 495, 498, 501, 504, 507, 510, 513, 516, 519,522, 525 or 528; and (iii) a VL CDR3 comprising the sequence shown inSEQ ID NO: 195, 198, 201, 204, 207, 210, 213, 216, 219, 222, 225, 228,231, 234, 237, 240, 243, 246, 249, 252, 255, 258, 261, 264, 466, 469,472, 475, 478, 481, 484, 487, 490, 493, 496, 499, 502, 505, 508, 511,514, 517, 520, 523, 526 or 529.

In some embodiments, the second antibody variable domain comprises theVH or VL region specific against CD3. For example, the second antibodyvariable domain comprises a heavy chain variable (VH) region comprisinga VH CDR1, VH CDR2, and VH CDR3 of the VH sequence shown in SEQ IDNO:266; or a light chain variable (VL) region comprising a VL CDR1, VLCDR2, and VL CDR3 of the VL sequence shown in SEQ ID NO: 265. In someembodiments, the second antibody variable domain comprises (a) a VHregion comprising (i) a VH CDR1 comprising the sequence shown in SEQ IDNO: 267, 268, or 269; (ii) a VH CDR2 comprising the sequence shown inSEQ ID NO: 270 or 271; and iii) a VH CDR3 comprising the sequence shownin SEQ ID NO: 272; or a VL region comprising (i) a VL CDR1 comprisingthe sequence shown in SEQ ID NO: 273; (ii) a VL CDR2 comprising thesequence shown in SEQ ID NO: 274; and (iii) a VL CDR3 comprising thesequence shown in SEQ ID NO: 275.

In some embodiments, the antibodies described herein comprise a constantregion. In some embodiments, the antibodies described herein are of thehuman IgG1, IgG2 or IgG2Δa, IgG3, or IgG4 subclass. In some embodiments,the antibodies described herein comprise a glycosylated constant region.In some embodiments, the antibodies described herein comprise a constantregion having decreased binding affinity to one or more human Fc gammareceptor(s).

In some embodiments, both the first and the second antibody variabledomains of the bispecific antibody comprise amino acid modifications atpositions 223, 225, and 228 (e.g., (C223E or C223R), (E225R), and (P228Eor P228R)) in the hinge region and at position 409 or 368 (e.g., K409Ror L368E (EU numbering scheme)) in the CH3 region of human IgG2 (SEQ IDNO: 279).

In some embodiments, both the first and the second antibody variabledomains of the bispecific antibody comprise amino acid modifications atposition 265 (e.g., D265A) of the human IgG2.

In some embodiments, both the first and the second antibody variabledomains of the bispecific antibody comprise amino acid modifications atone or more of positions 265 (e.g., D265A), 330 (e.g., A330S), and 331(e.g., P331S) of the human IgG2. In some embodiments, both the first andthe second antibody variable domains of the bispecific antibody compriseamino acid modifications at each of positions 265 (e.g., D265A), 330(e.g., A330S), and 331 (e.g., P331S) of the human IgG2.

In other embodiments, the invention provides pharmaceutical compositionscomprising any of the antibodies described herein.

The invention also provides cell lines that recombinantly produce any ofthe antibodies described herein.

The invention also provides nucleic acids encoding any of the antibodiesdescribed herein. The invention also provides nucleic acids encoding aheavy chain variable region or a light chain variable region of any ofthe antibodies described herein.

The invention also provides a host cell comprising a nucleic acid orvector provided herein. Also provided is a method of producing anantibody (e.g. monospecific or bispecific) provided herein, comprisingculturing a host cell provided herein under conditions that result inproduction of the antibody, and isolating the antibody from the hostcell or culture.

The invention also provides kits comprising an effective amount of anyof the antibodies or antibody conjugates described herein.

Also provided is an antibody or bispecific antibody provided herein foruse as a medicament.

The invention also provides methods of treating subjects in need thereofcomprising providing the isolated antibodies or bispecific antibodiesdescribed herein, and administering said antibodies to said subject.

Also provided are methods of treating a condition associated withmalignant cells expressing CD70 in a subject comprising administering toa subject in need thereof an effective amount of a pharmaceuticalcomposition comprising the antibodies as described herein. In someembodiments, the condition is a cancer. In some embodiments, the canceris an CD70 related cancer (e.g., any cancer with CD70 expression)selected from the group consisting of Renal Cell Carcinoma,Glioblastoma, glioma such as low grade glioma, Non-Hodgkin's Lymphoma(NHL), Hodgkin's Disease (HD), Waldenstrom's macroglobulinemia, AcuteMyeloid Leukemia, Multiple Myeloma, diffuse large-cell lymphoma,follicular lymphoma or Non-Small Cell Lung Cancer.

In another aspect, the invention provides a method of inhibiting tumorgrowth or progression in a subject who has malignant cells expressingCD70, comprising administering to the subject in need thereof aneffective amount of a pharmaceutical composition comprising the isolatedantibodies or bispecific antibodies, as described herein.

In another aspect, the invention provides a method of inhibitingmetastasis of malignant cells expressing CD70 in a subject, comprisingadministering to the subject in need thereof an effective amount of thepharmaceutical composition comprising the isolated antibodies orbispecific antibodies, as described herein.

In another aspect, the invention provides a method inducing tumorregression in a subject who has malignant cells expressing CD70,comprising administering to the subject in need thereof an effectiveamount of the pharmaceutical composition of a pharmaceutical compositioncomprising the isolated antibodies or bispecific antibodies, asdescribed herein.

DETAILED DESCRIPTION

The invention disclosed herein provides antibodies (e.g., monospecificor bispecific) that specifically bind to CD70 (e.g., human CD70). Theinvention also provides polynucleotides encoding these antibodies,compositions comprising these antibodies, and methods of making andusing these antibodies. The invention also provides methods for treatinga condition associated with CD70-mediated pathologies in a subject, suchas cancer. In particular, the inventors of the present invention havediscovered that the CD70 antibodies as described herein in thefull-length bispecific format have longer half-life, minimizedFc-interaction, and minimized non-specific cytokine release in vivo viainteraction with the immune cells.

General Techniques

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology (includingrecombinant techniques), microbiology, cell biology, biochemistry,immunology, virology, monoclonal antibody generation and engineering,which are within the skill of the art. Such techniques are explainedfully in the literature, such as, Molecular Cloning: A LaboratoryManual, second edition (Sambrook et al., 1989) Cold Spring Harbor Press;Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Methods in MolecularBiology, Humana Press; Cell Biology: A Laboratory Notebook (J. E.Cellis, ed., 1998) Academic Press; Animal Cell Culture (R. I. Freshney,ed., 1987); Introduction to Cell and Tissue Culture (J. P. Mather and P.E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: LaboratoryProcedures (A. Doyle, J. B. Griffiths, and D. G. Newell, eds.,1993-1998) J. Wiley and Sons; Methods in Enzymology (Academic Press,Inc.); Handbook of Experimental Immunology (D. M. Weir and C. C.Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J. M.Miller and M. P. Calos, eds., 1987); Current Protocols in MolecularBiology (F. M. Ausubel et al., eds., 1987); PCR: The Polymerase ChainReaction, (Mullis et al., eds., 1994); Current Protocols in Immunology(J. E. Coligan et al., eds., 1991); Short Protocols in Molecular Biology(Wiley and Sons, 1999); Immunobiology (C. A. Janeway and P. Travers,1997); Antibodies (P. Finch, 1997); Antibodies: a practical approach (D.Catty, ed., IRL Press, 1988-1989); Monoclonal antibodies: a practicalapproach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000);Using antibodies: a laboratory manual (E. Harlow and D. Lane (ColdSpring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J.D. Capra, eds., Harwood Academic Publishers, 1995).

Definitions

An “antibody” is an immunoglobulin molecule capable of specific bindingto a target, such as a carbohydrate, polynucleotide, lipid, polypeptide,etc., through at least one antigen recognition site, located in thevariable region of the immunoglobulin molecule. As used herein, the termencompasses not only intact polyclonal or monoclonal antibodies, butalso antigen binding fragments thereof (such as Fab, Fab′, F(ab′)2, Fv),single chain (ScFv) and domain antibodies (including, for example, sharkand camelid antibodies), and fusion proteins comprising an antibody, andany other modified configuration of the immunoglobulin molecule thatcomprises an antigen recognition site. An antibody includes an antibodyof any class, such as IgG, IgA, or IgM (or sub-class thereof), and theantibody need not be of any particular class. Depending on the antibodyamino acid sequence of the constant region of its heavy chains,immunoglobulins can be assigned to different classes. There are fivemajor classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, andseveral of these may be further divided into subclasses (isotypes),e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. The heavy-chain constantregions that correspond to the different classes of immunoglobulins arecalled alpha, delta, epsilon, gamma, and mu, respectively. The subunitstructures and three-dimensional configurations of different classes ofimmunoglobulins are well known.

The term “antigen binding fragment” or “antigen binding portion” of anantibody, as used herein, refers to one or more fragments of an intactantibody that retain the ability to specifically bind to a given antigen(e.g., CD70). Antigen binding functions of an antibody can be performedby fragments of an intact antibody. Examples of binding fragmentsencompassed within the term “antigen binding fragment” of an antibodyinclude Fab; Fab′; F(ab′)2; an Fd fragment consisting of the VH and CH1domains; an Fv fragment consisting of the VL and VH domains of a singlearm of an antibody; a single domain antibody (dAb) fragment (Ward etal., Nature 341:544-546, 1989), and an isolated complementaritydetermining region (CDR).

An antibody or a polypeptide that “preferentially binds” or“specifically binds” (used interchangeably herein) to a target (e.g.,CD70 protein) is a term well understood in the art, and methods todetermine such specific or preferential binding are also well known inthe art. A molecule is said to exhibit “specific binding” or“preferential binding” if it reacts or associates more frequently, morerapidly, with greater duration or with greater affinity with aparticular cell or substance than it does with alternative cells orsubstances. An antibody “specifically binds” or “preferentially binds”to a target if it binds with greater affinity, avidity, more readily, orwith greater duration than it binds to other substances. For example, anantibody that specifically or preferentially binds to an CD70 epitope isan antibody that binds this epitope with greater affinity, avidity, morereadily, or with greater duration than it binds to other CD70 epitopesor non-CD70 epitopes. It is also understood that by reading thisdefinition, for example, an antibody (or moiety or epitope) thatspecifically or preferentially binds to a first target may or may notspecifically or preferentially bind to a second target. As such,“specific binding” or “preferential binding” does not necessarilyrequire (although it can include) exclusive binding. Generally, but notnecessarily, reference to binding means preferential binding.

A “variable region” of an antibody refers to the variable region of theantibody light chain or the variable region of the antibody heavy chain,either alone or in combination. As known in the art, the variableregions of the heavy and light chain each consist of four frameworkregions (FR) connected by three complementarity determining regions(CDRs) also known as hypervariable regions. The CDRs in each chain areheld together in close proximity by the FRs and, with the CDRs from theother chain, contribute to the formation of the antigen binding site ofantibodies. There are at least two techniques for determining CDRs: (1)an approach based on cross-species sequence variability (i.e., Kabat etal. Sequences of Proteins of Immunological Interest, (5th ed., 1991,National Institutes of Health, Bethesda Md.)); and (2) an approach basedon crystallographic studies of antigen-antibody complexes (Al-lazikaniet al., 1997, J. Molec. Biol. 273:927-948). As used herein, a CDR mayrefer to CDRs defined by either approach or by a combination of bothapproaches.

A “CDR” of a variable domain are amino acid residues within the variableregion that are identified in accordance with the definitions of theKabat, Chothia, the accumulation of both Kabat and Chothia, AbM,contact, or conformational definitions or any method of CDRdetermination well known in the art. Antibody CDRs may be identified asthe hypervariable regions originally defined by Kabat et al. See, e.g.,Kabat et al., 1992, Sequences of Proteins of Immunological Interest, 5thed., Public Health Service, NIH, Washington D.C. The positions of theCDRs may also be identified as the structural loop structures originallydescribed by Chothia and others. See, e.g., Chothia et al., Nature342:877-883, 1989. Other approaches to CDR identification include the“AbM definition,” which is a compromise between Kabat and Chothia and isderived using Oxford Molecular's AbM antibody modeling software (nowAccelrys®), or the “contact definition” of CDRs based on observedantigen contacts, set forth in MacCallum et al., J. Mol. Biol.,262:732-745, 1996. In another approach, referred to herein as the“conformational definition” of CDRs, the positions of the CDRs may beidentified as the residues that make enthalpic contributions to antigenbinding. See, e.g., Makabe et al., Journal of Biological Chemistry,283:1156-1166, 2008. Still other CDR boundary definitions may notstrictly follow one of the above approaches, but will nonethelessoverlap with at least a portion of the Kabat CDRs, although they may beshortened or lengthened in light of prediction or experimental findingsthat particular residues or groups of residues or even entire CDRs donot significantly impact antigen binding. As used herein, a CDR mayrefer to CDRs defined by any approach known in the art, includingcombinations of approaches. The methods used herein may utilize CDRsdefined according to any of these approaches. For any given embodimentcontaining more than one CDR, the CDRs may be defined in accordance withany of Kabat, Chothia, extended, AbM, contact, or conformationaldefinitions.

As used herein, “monoclonal antibody” refers to an antibody obtainedfrom a population of substantially homogeneous antibodies, i.e., theindividual antibodies comprising the population are identical except forpossible naturally-occurring mutations that may be present in minoramounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast to polyclonalantibody preparations, which typically include different antibodiesdirected against different determinants (epitopes), each monoclonalantibody is directed against a single determinant on the antigen. Themodifier “monoclonal” indicates the character of the antibody as beingobtained from a substantially homogeneous population of antibodies, andis not to be construed as requiring production of the antibody by anyparticular method. For example, the monoclonal antibodies to be used inaccordance with the present invention may be made by the hybridomamethod first described by Kohler and Milstein, Nature 256:495, 1975, ormay be made by recombinant DNA methods such as described in U.S. Pat.No. 4,816,567. The monoclonal antibodies may also be isolated from phagelibraries generated using the techniques described in McCafferty et al.,Nature 348:552-554, 1990, for example.

As used herein, “humanized” antibody refers to forms of non-human (e.g.murine) antibodies that are chimeric immunoglobulins, immunoglobulinchains, or fragments thereof (such as Fv, Fab, Fab′, F(ab′)2 or otherantigen binding subsequences of antibodies) that contain minimalsequence derived from non-human immunoglobulin.

Preferably, humanized antibodies are human immunoglobulins (recipientantibody) in which residues from a complementary determining region(CDR) of the recipient are replaced by residues from a CDR of anon-human species (donor antibody) such as mouse, rat, or rabbit havingthe desired specificity, affinity, and capacity. In some instances, Fvframework region (FR) residues of the human immunoglobulin are replacedby corresponding non-human residues. Furthermore, the humanized antibodymay comprise residues that are found neither in the recipient antibodynor in the imported CDR or framework sequences, but are included tofurther refine and optimize antibody performance. In general, thehumanized antibody will comprise substantially all of at least one, andtypically two, variable domains, in which all or substantially all ofthe CDR regions correspond to those of a non-human immunoglobulin andall or substantially all of the FR regions are those of a humanimmunoglobulin consensus sequence. The humanized antibody optimally alsowill comprise at least a portion of an immunoglobulin constant region ordomain (Fc), typically that of a human immunoglobulin. Preferred areantibodies having Fc regions modified as described in WO 99/58572. Otherforms of humanized antibodies have one or more CDRs (CDR L1, CDR L2, CDRL3, CDR H1, CDR H2, or CDR H3) which are altered with respect to theoriginal antibody, which are also termed one or more CDRs “derived from”one or more CDRs from the original antibody.

As used herein, “human antibody” means an antibody having an amino acidsequence corresponding to that of an antibody produced by a human orwhich has been made using any of the techniques for making humanantibodies known to those skilled in the art or disclosed herein. Thisdefinition of a human antibody includes antibodies comprising at leastone human heavy chain polypeptide or at least one human light chainpolypeptide. One such example is an antibody comprising murine lightchain and human heavy chain polypeptides. Human antibodies can beproduced using various techniques known in the art. In one embodiment,the human antibody is selected from a phage library, where that phagelibrary expresses human antibodies (Vaughan et al., NatureBiotechnology, 14:309-314, 1996; Sheets et al., Proc. Natl. Acad. Sci.(USA) 95:6157-6162, 1998; Hoogenboom and Winter, J. Mol. Biol., 227:381,1991; Marks et al., J. Mol. Biol., 222:581, 1991). Human antibodies canalso be made by immunization of animals into which human immunoglobulinloci have been transgenically introduced in place of the endogenousloci, e.g., mice in which the endogenous immunoglobulin genes have beenpartially or completely inactivated. This approach is described in U.S.Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and5,661,016. Alternatively, the human antibody may be prepared byimmortalizing human B lymphocytes that produce an antibody directedagainst a target antigen (such B lymphocytes may be recovered from anindividual or from single cell cloning of the cDNA, or may have beenimmunized in vitro). See, e.g., Cole et al. Monoclonal Antibodies andCancer Therapy, Alan R. Liss, p. 77, 1985; Boerner et al., J. Immunol.,147 (1):86-95, 1991; and U.S. Pat. No. 5,750,373.

The term “chimeric antibody” is intended to refer to antibodies in whichthe variable region sequences are derived from one species and theconstant region sequences are derived from another species, such as anantibody in which the variable region sequences are derived from a mouseantibody and the constant region sequences are derived from a humanantibody.

The terms “polypeptide”, “oligopeptide”, “peptide” and “protein” areused interchangeably herein to refer to chains of amino acids of anylength. For example, the chain may be relatively short (e.g., 10-100amino acids), or longer. The chain may be linear or branched, it maycomprise modified amino acids, or may be interrupted by non-amino acids.The terms also encompass an amino acid chain that has been modifiednaturally or by intervention; for example, disulfide bond formation,glycosylation, lipidation, acetylation, phosphorylation, or any othermanipulation or modification, such as conjugation with a labelingcomponent. Also included within the definition are, for example,polypeptides containing one or more analogs of an amino acid (including,for example, unnatural amino acids, etc.), as well as othermodifications known in the art. It is understood that the polypeptidescan occur as single chains or associated chains.

A “monovalent antibody” comprises one antigen binding site per molecule(e.g., IgG or Fab). In some instances, a monovalent antibody can havemore than one antigen binding sites, but the binding sites are fromdifferent antigens.

A “monospecific antibody” comprises two identical antigen binding sitesper molecule (e.g. IgG) such that the two binding sites bind identicalepitope on the antigen.

Thus, they compete with each other on binding to one antigen molecule.Most antibodies found in nature are monospecific. In some instances, amonospecific antibody can also be a monovalent antibody (e.g. Fab)

A “bivalent antibody” comprises two antigen binding sites per molecule(e.g., IgG). In some instances, the two binding sites have the sameantigen specificities. However, bivalent antibodies may be bispecific.

A “bispecific” or “dual-specific” is a hybrid antibody having twodifferent antigen binding sites. The two antigen binding sites of abispecific antibody bind to two different epitopes, which may reside onthe same or different protein targets.

A “bifunctional” is antibody is an antibody having identical antigenbinding sites (i.e., identical amino acid sequences) in the two arms buteach binding site can recognize two different antigens.

A “heteromultimer”, “heteromultimeric complex”, or “heteromultimericpolypeptide” is a molecule comprising at least a first polypeptide and asecond polypeptide, wherein the second polypeptide differs in amino acidsequence from the first polypeptide by at least one amino acid residue.The heteromultimer can comprise a “heterodimer” formed by the first andsecond polypeptide or can form higher order tertiary structures wherepolypeptides in addition to the first and second polypeptide arepresent.

A “heterodimer,” “heterodimeric protein,” “heterodimeric complex,” or“heteromultimeric polypeptide” is a molecule comprising a firstpolypeptide and a second polypeptide, wherein the second polypeptidediffers in amino acid sequence from the first polypeptide by at leastone amino acid residue.

The “hinge region,” “hinge sequence”, and variations thereof, as usedherein, includes the meaning known in the art, which is illustrated in,for example, Janeway et al., ImmunoBiology: the immune system in healthand disease, (Elsevier Science Ltd., NY) (4th ed., 1999); Bloom et al.,Protein Science (1997), 6:407-415; Humphreys et al., J. Immunol. Methods(1997), 209:193-202.

The “immunoglobulin-like hinge region,” “immunoglobulin-like hingesequence,” and variations thereof, as used herein, refer to the hingeregion and hinge sequence of an immunoglobulin-like or an antibody-likemolecule (e.g., immunoadhesins). In some embodiments, theimmunoglobulin-like hinge region can be from or derived from any IgG1,IgG2, IgG3, or IgG4 subtype, or from IgA, IgE, IgD or IgM, includingchimeric forms thereof, e.g., a chimeric IgG1/2 hinge region.

The term “immune effector cell” or “effector cell as used herein refersto a cell within the natural repertoire of cells in the human immunesystem which can be activated to affect the viability of a target cell.The viability of a target cell can include cell survival, proliferation,or ability to interact with other cells.

Antibodies of the invention can be produced using techniques well knownin the art, e.g., recombinant technologies, phage display technologies,synthetic technologies or combinations of such technologies or othertechnologies readily known in the art (see, for example, Jayasena, S.D., Clin. Chem., 45: 1628-50, 1999 and Fellouse, F. A., et al, J. Mol.Biol., 373(4):924-40, 2007).

As known in the art, “polynucleotide,” or “nucleic acid,” as usedinterchangeably herein, refer to chains of nucleotides of any length,and include DNA and RNA. The nucleotides can be deoxyribonucleotides,ribonucleotides, modified nucleotides or bases, or their analogs, or anysubstrate that can be incorporated into a chain by DNA or RNApolymerase. A polynucleotide may comprise modified nucleotides, such asmethylated nucleotides and their analogs. If present, modification tothe nucleotide structure may be imparted before or after assembly of thechain. The sequence of nucleotides may be interrupted by non-nucleotidecomponents. A polynucleotide may be further modified afterpolymerization, such as by conjugation with a labeling component. Othertypes of modifications include, for example, “caps”, substitution of oneor more of the naturally occurring nucleotides with an analog,internucleotide modifications such as, for example, those with unchargedlinkages (e.g., methyl phosphonates, phosphotriesters, phosphoamidates,carbamates, etc.) and with charged linkages (e.g., phosphorothioates,phosphorodithioates, etc.), those containing pendant moieties, such as,for example, proteins (e.g., nucleases, toxins, antibodies, signalpeptides, poly-L-lysine, etc.), those with intercalators (e.g.,acridine, psoralen, etc.), those containing chelators (e.g., metals,radioactive metals, boron, oxidative metals, etc.), those containingalkylators, those with modified linkages (e.g., alpha anomeric nucleicacids, etc.), as well as unmodified forms of the polynucleotide(s).Further, any of the hydroxyl groups ordinarily present in the sugars maybe replaced, for example, by phosphonate groups, phosphate groups,protected by standard protecting groups, or activated to prepareadditional linkages to additional nucleotides, or may be conjugated tosolid supports. The 5′ and 3′ terminal OH can be phosphorylated orsubstituted with amines or organic capping group moieties of from 1 to20 carbon atoms. Other hydroxyls may also be derivatized to standardprotecting groups. Polynucleotides can also contain analogous forms ofribose or deoxyribose sugars that are generally known in the art,including, for example, 2′-O-methyl-, 2′-O-allyl, 2′-fluoro- or2′-azido-ribose, carbocyclic sugar analogs, alpha- or beta-anomericsugars, epimeric sugars such as arabinose, xyloses or lyxoses, pyranosesugars, furanose sugars, sedoheptuloses, acyclic analogs and abasicnucleoside analogs such as methyl riboside. One or more phosphodiesterlinkages may be replaced by alternative linking groups. Thesealternative linking groups include, but are not limited to, embodimentswherein phosphate is replaced by P(O)S(“thioate”), P(S)S (“dithioate”),(O)NR₂ (“am idate”), P(O)R, P(O)OR′, CO or CH2 (“formacetal”), in whicheach R or R′ is independently H or substituted or unsubstituted alkyl(1-20 C) optionally containing an ether (—O—) linkage, aryl, alkenyl,cycloalkyl, cycloalkenyl or araldyl. Not all linkages in apolynucleotide need be identical. The preceding description applies toall polynucleotides referred to herein, including RNA and DNA.

As known in the art, a “constant region” of an antibody refers to theconstant region of the antibody light chain or the constant region ofthe antibody heavy chain, either alone or in combination.

As used herein, “substantially pure” refers to material which is atleast 50% pure (i.e., free from contaminants), more preferably, at least90% pure, more preferably, at least 95% pure, yet more preferably, atleast 98% pure, and most preferably, at least 99% pure.

A “host cell” includes an individual cell or cell culture that can be orhas been a recipient for vector(s) for incorporation of polynucleotideinserts. Host cells include progeny of a single host cell, and theprogeny may not necessarily be completely identical (in morphology or ingenomic DNA complement) to the original parent cell due to natural,accidental, or deliberate mutation. A host cell includes cellstransfected in vivo with a polynucleotide(s) of this invention.

As known in the art, the term “Fc region” is used to define a C-terminalregion of an immunoglobulin heavy chain. The “Fc region” may be a nativesequence Fc region or a variant Fc region. Although the boundaries ofthe Fc region of an immunoglobulin heavy chain might vary, the human IgGheavy chain Fc region is usually defined to stretch from an amino acidresidue at position Cys226, or from Pro230, to the carboxyl-terminusthereof. The numbering of the residues in the Fc region is that of theEU index as in Kabat. Kabat et al., Sequences of Proteins ofImmunological Interest, 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md., 1991. The Fc region of animmunoglobulin generally comprises two constant regions, CH2 and CH3.

As used in the art, “Fc receptor” and “FcR” describe a receptor thatbinds to the Fc region of an antibody. The preferred FcR is a nativesequence human FcR. Moreover, a preferred FcR is one which binds an IgGantibody (a gamma receptor) and includes receptors of the FcγRI, FcγRII,and FcγRIII subclasses, including allelic variants and alternativelyspliced forms of these receptors. FcγRII receptors include FcγRIIA (an“activating receptor”) and FcγRIIB (an “inhibiting receptor”), whichhave similar amino acid sequences that differ primarily in thecytoplasmic domains thereof. FcRs are reviewed in Ravetch and Kinet,Ann. Rev. Immunol., 9:457-92, 1991; Capel et al., Immunomethods,4:25-34, 1994; and de Haas et al., J. Lab. Clin. Med., 126:330-41, 1995.“FcR” also includes the neonatal receptor, FcRn, which is responsiblefor the transfer of maternal IgGs to the fetus (Guyer et al., J.Immunol., 117:587, 1976; and Kim et al., J. Immunol., 24:249, 1994).

The term “compete”, as used herein with regard to an antibody, meansthat a first antibody, or an antigen binding fragment (or portion)thereof, binds to an epitope in a manner sufficiently similar to thebinding of a second antibody, or an antigen binding portion thereof,such that the result of binding of the first antibody with its cognateepitope is detectably decreased in the presence of the second antibodycompared to the binding of the first antibody in the absence of thesecond antibody. The alternative, where the binding of the secondantibody to its epitope is also detectably decreased in the presence ofthe first antibody, can, but need not be the case. That is, a firstantibody can inhibit the binding of a second antibody to its epitopewithout that second antibody inhibiting the binding of the firstantibody to its respective epitope. However, where each antibodydetectably inhibits the binding of the other antibody with its cognateepitope or ligand, whether to the same, greater, or lesser extent, theantibodies are said to “cross-compete” with each other for binding oftheir respective epitope(s). Both competing and cross-competingantibodies are encompassed by the present invention. Regardless of themechanism by which such competition or cross-competition occurs (e.g.,steric hindrance, conformational change, or binding to a common epitope,or portion thereof), the skilled artisan would appreciate, based uponthe teachings provided herein, that such competing or cross-competingantibodies are encompassed and can be useful for the methods disclosedherein.

A “functional Fc region” possesses at least one effector function of anative sequence Fc region. Exemplary “effector functions” include C1qbinding; complement dependent cytotoxicity; Fc receptor binding;antibody-dependent cell-mediated cytotoxicity; phagocytosis;down-regulation of cell surface receptors (e.g. B cell receptor), etc.Such effector functions generally require the Fc region to be combinedwith a binding domain (e.g. an antibody variable domain) and can beassessed using various assays known in the art for evaluating suchantibody effector functions.

A “native sequence Fc region” comprises an amino acid sequence identicalto the amino acid sequence of an Fc region found in nature. A “variantFc region” comprises an amino acid sequence which differs from that of anative sequence Fc region by virtue of at least one amino acidmodification, yet retains at least one effector function of the nativesequence Fc region. In some embodiments, the variant Fc region has atleast one amino acid substitution compared to a native sequence Fcregion or to the Fc region of a parent polypeptide, e.g. from about oneto about ten amino acid substitutions, and preferably, from about one toabout five amino acid substitutions in a native sequence Fc region or inthe Fc region of the parent polypeptide. The variant Fc region hereinwill preferably possess at least about 80% sequence identity with anative sequence Fc region or with an Fc region of a parent polypeptide,and most preferably, at least about 90% sequence identity therewith,more preferably, at least about 95%, at least about 96%, at least about97%, at least about 98%, at least about 99% sequence identity therewith.

The term “effector function” refers to the biological activitiesattributable to the Fc region of an antibody. Examples of antibodyeffector functions include, but are not limited to, antibody-dependentcell-mediated cytotoxicity (ADCC), Fc receptor binding, complementdependent cytotoxicity (CDC), phagocytosis, C1q binding, and downregulation of cell surface receptors (e.g., B cell receptor; BCR). See,e.g., U.S. Pat. No. 6,737,056. Such effector functions generally requirethe Fc region to be combined with a binding domain (e.g., an antibodyvariable domain) and can be assessed using various assays known in theart for evaluating such antibody effector functions. An exemplarymeasurement of effector function is through Fcγ3 or C1q binding.

As used herein “antibody-dependent cell-mediated cytotoxicity” or “ADCC”refers to a cell-mediated reaction in which nonspecific cytotoxic cellsthat express Fc receptors (FcRs) (e.g. natural killer (NK) cells,neutrophils, and macrophages) recognize bound antibody on a target celland subsequently cause lysis of the target cell. ADCC activity of amolecule of interest can be assessed using an in vitro ADCC assay, suchas that described in U.S. Pat. No. 5,500,362 or 5,821,337. Usefuleffector cells for such assays include peripheral blood mononuclearcells (PBMC) and NK cells. Alternatively, or additionally, ADCC activityof the molecule of interest may be assessed in vivo, e.g., in an animalmodel such as that disclosed in Clynes et al., 1998, PNAS (USA),95:652-656.

“Complement dependent cytotoxicity” or “CDC” refers to the lysing of atarget in the presence of complement. The complement activation pathwayis initiated by the binding of the first component of the complementsystem (C1q) to a molecule (e.g. an antibody) complexed with a cognateantigen. To assess complement activation, a CDC assay, e.g. as describedin Gazzano-Santoro et al., J. Immunol. Methods, 202: 163 (1996), may beperformed.

As used herein, “treatment” is an approach for obtaining beneficial ordesired clinical results. For purposes of this invention, beneficial ordesired clinical results include, but are not limited to, one or more ofthe following: reducing the proliferation of (or destroying) neoplasticor cancerous cells, inhibiting metastasis of neoplastic cells, shrinkingor decreasing the size of CD70 expressing tumor, remission of an CD70associated disease (e.g., cancer), decreasing symptoms resulting from anCD70 associated disease (e.g., cancer), increasing the quality of lifeof those suffering from an CD70 associated disease (e.g., cancer),decreasing the dose of other medications required to treat an CD70associated disease (e.g., cancer), delaying the progression of an CD70associated disease (e.g., cancer), curing an CD70 associated disease(e.g., cancer), or prolong survival of patients having an CD70associated disease (e.g., cancer).

“Ameliorating” means a lessening or improvement of one or more symptomsas compared to not administering an CD70 antibody (monospecific orbispecific). “Ameliorating” also includes shortening or reduction induration of a symptom.

As used herein, an “effective dosage” or “effective amount” of drug,compound, or pharmaceutical composition is an amount sufficient toeffect any one or more beneficial or desired results. For prophylacticuse, beneficial or desired results include eliminating or reducing therisk, lessening the severity, or delaying the outset of the disease,including biochemical, histological or behavioral symptoms of thedisease, its complications and intermediate pathological phenotypespresenting during development of the disease. For therapeutic use,beneficial or desired results include clinical results such as reducingincidence or amelioration of one or more symptoms of various CD70associated diseases or conditions (such as for example multiplemyeloma), decreasing the dose of other medications required to treat thedisease, enhancing the effect of another medication, or delaying theprogression of the CD70 associated disease of patients. An effectivedosage can be administered in one or more administrations. For purposesof this invention, an effective dosage of drug, compound, orpharmaceutical composition is an amount sufficient to accomplishprophylactic or therapeutic treatment either directly or indirectly. Asis understood in the clinical context, an effective dosage of a drug,compound, or pharmaceutical composition may or may not be achieved inconjunction with another drug, compound, or pharmaceutical composition.Thus, an “effective dosage” may be considered in the context ofadministering one or more therapeutic agents, and a single agent may beconsidered to be given in an effective amount if, in conjunction withone or more other agents, a desirable result may be or is achieved.

An “individual” or a “subject” is a mammal, more preferably, a human.Mammals also include, but are not limited to primates, horses, dogs,cats, mice and rats.

As used herein, “vector” means a construct, which is capable ofdelivering, and, preferably, expressing, one or more gene(s) orsequence(s) of interest in a host cell. Examples of vectors include, butare not limited to, viral vectors, naked DNA or RNA expression vectors,plasmid, cosmid or phage vectors, DNA or RNA expression vectorsassociated with cationic condensing agents, DNA or RNA expressionvectors encapsulated in liposomes, and certain eukaryotic cells, such asproducer cells.

As used herein, “expression control sequence” means a nucleic acidsequence that directs transcription of a nucleic acid. An expressioncontrol sequence can be a promoter, such as a constitutive or aninducible promoter, or an enhancer. The expression control sequence isoperably linked to the nucleic acid sequence to be transcribed.

As used herein, “pharmaceutically acceptable carrier” or “pharmaceuticalacceptable excipient” includes any material which, when combined with anactive ingredient, allows the ingredient to retain biological activityand is non-reactive with the subject's immune system. Examples include,but are not limited to, any of the standard pharmaceutical carriers suchas a phosphate buffered saline solution, water, emulsions such asoil/water emulsion, and various types of wetting agents. Preferreddiluents for aerosol or parenteral administration are phosphate bufferedsaline (PBS) or normal (0.9%) saline. Compositions comprising suchcarriers are formulated by well known conventional methods (see, forexample, Remington's Pharmaceutical Sciences, 18th edition, A. Gennaro,ed., Mack Publishing Co., Easton, Pa., 1990; and Remington, The Scienceand Practice of Pharmacy 21st Ed. Mack Publishing, 2005).

The term “acyl donor glutamine-containing tag” or “glutamine tag” asused herein refers to a polypeptide or a protein containing one or moreGln residue(s) that acts as a transglutaminase amine acceptor. See,e.g., WO2012059882 and WO2015015448.

The term “k_(on)” or “k_(a)”, as used herein, refers to the rateconstant for association of an antibody to an antigen. Specifically, therate constants (k_(on)/k_(a) and k_(off)/k_(d)) and equilibriumdissociation constants are measured using whole antibody (i.e. bivalent)and monomeric CD70 proteins (e.g., Histidine-tagged CD70 fusionprotein).

The term “k_(off)” or “k_(d)”, as used herein, refers to the rateconstant for dissociation of an antibody from the antibody/antigencomplex.

The term “K_(D)”, as used herein, refers to the equilibrium dissociationconstant of an antibody-antigen interaction.

Reference to “about” a value or parameter herein includes (anddescribes) embodiments that are directed to that value or parameter perse. For example, description referring to “about X” includes descriptionof “X.” Numeric ranges are inclusive of the numbers defining the range.Generally speaking, the term “about” refers to the indicated value ofthe variable and to all values of the variable that are within theexperimental error of the indicated value (e.g. within the 95%confidence interval for the mean) or within 10 percent of the indicatedvalue, whichever is greater. Where the term “about” is used within thecontext of a time period (years, months, weeks, days etc.), the term“about” means that period of time plus or minus one amount of the nextsubordinate time period (e.g. about 1 year means 11-13 months; about 6months means 6 months plus or minus 1 week; about 1 week means 6-8 days;etc.), or within 10 percent of the indicated value, whichever isgreater.

It is understood that wherever embodiments are described herein with thelanguage “comprising,” otherwise analogous embodiments described interms of “consisting of” or “consisting essentially of” are alsoprovided.

Where aspects or embodiments of the invention are described in terms ofa Markush group or other grouping of alternatives, the present inventionencompasses not only the entire group listed as a whole, but each memberof the group individually and all possible subgroups of the main group,but also the main group absent one or more of the group members. Thepresent invention also envisages the explicit exclusion of one or moreof any of the group members in the claimed invention.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. In case of conflict, thepresent specification, including definitions, will control. Throughoutthis specification and claims, the word “comprise,” or variations suchas “comprises” or “comprising” will be understood to imply the inclusionof a stated integer or group of integers but not the exclusion of anyother integer or group of integers. Unless otherwise required bycontext, singular terms shall include pluralities and plural terms shallinclude the singular.

Exemplary methods and materials are described herein, although methodsand materials similar or equivalent to those described herein can alsobe used in the practice or testing of the present invention. Thematerials, methods, and examples are illustrative only and not intendedto be limiting.

CD70 Antibodies and Methods of Making Thereof

The present invention provides an antibody that binds to CD70 [e.g.,human CD70 (e.g., accession number: NP_004110 or SEQ ID NO: 235)] andcharacterized by any one or more of the following characteristics: (a)treat, prevent, ameliorate one or more symptoms of a conditionassociated with malignant cells expressing CD70 in a subject (e.g.,cancer such as AML); (b) inhibit tumor growth or progression in asubject (who has a malignant tumor expressing CD70); (c) inhibitmetastasis of cancer (malignant) cells expressing CD70 in a subject (whohas one or more malignant cells expressing CD70); (d) induce regression(e.g., long-term regression) of a tumor expressing CD70; (e) exertcytotoxic activity in malignant cells expressing CD70; (f) block CD70interaction with other yet to be identified factors; or (g) inducebystander effect that kill or inhibit growth of non-CD70 expressingmalignant cells in the vicinity.

In one aspect, provided is an isolated antibody which specifically bindsto CD70, wherein the antibody comprises (a) a heavy chain variable (VH)region comprising (i) a VH complementarity determining region one (CDR1)comprising the sequence shown in 49, 50, 51, 55, 56, 57, 61, 62, 63, 67,68, 69, 73, 74, 75, 79, 80, 81, 85, 86, 87, 91, 92, 93, 97, 98, 99, 103,104, 105, 109, 110, 111, 115, 116, 117, 121, 122, 123, 127, 128, 129,133, 134, 135, 139, 140, 141, 145, 146, 147, 151, 152, 153, 157, 158,159, 163, 164, 165, 169, 170, 171, 175, 176, 177, 181, 182, 183, 187,188, 189, 332, 333, 334, 338, 339, 340, 344, 345, 346, 350, 351, 352,356, 357, 358, 362, 363, 364, 368, 369, 370, 374, 375, 376, 380, 381,382, 386, 387, 388, 392, 393, 394, 398, 399, 400, 404, 405, 406, 410,411, 412, 416, 437, 418, 422, 423, 424, 428, 429, 430, 434, 435, 436,440, 441, 442, 446, 447, 448, 452, 453, 454, 458, 459 or 460; (ii) a VHCDR2 comprising the sequence shown in SEQ ID NO: 52, 53, 58, 59, 64, 65,70, 71, 76, 77, 82, 83, 88, 89, 94, 95, 100, 101, 106, 107, 112, 113,118, 119, 124, 125, 130, 131, 136, 137, 142, 143, 148, 149, 154, 155,160, 161, 166, 167, 172, 173, 178, 179, 184, 185, 190, 191, 335, 336,341, 342, 347, 348, 353, 354, 359, 360, 365, 366, 371, 372, 377, 378,383, 384, 389, 390, 395, 396, 401, 402, 407, 408, 413, 414, 419, 420,425, 426, 431, 432, 437, 438, 443, 444, 449, 450, 455, 456, 461 or 462;and iii) a VH CDR3 comprising the sequence shown in SEQ ID NO: 54, 60,66, 72, 78, 84, 90, 96, 102, 108, 114, 120, 126, 132, 138, 144, 150,156, 162, 168, 174, 180, 186, 192, 337, 343, 349, 355, 361, 367, 373,379, 385, 391, 397, 403, 409, 415, 421, 427, 433, 439, 445, 451, 457 or463; or a light chain variable (VL) region comprising (i) a VL CDR1comprising the sequence shown in SEQ ID NO: 193, 196, 199, 202, 205,208, 211, 214, 217, 220, 223, 226, 229, 232, 235, 238, 241, 244, 247,250, 253, 256, 259, 262, 464, 467, 470, 473, 476, 479, 482, 485, 488,491, 494, 497, 500, 503, 506, 509, 512, 515, 518, 521, 524 or 527; (ii)a VL CDR2 comprising the sequence shown in SEQ ID NO: 194, 197, 200,203, 206, 209, 212, 215, 218, 221, 224, 227, 230, 233, 236, 239, 242,245, 248, 251, 254, 257, 260, 263, 465, 468, 471, 474, 477, 480, 483,486, 489, 492, 495, 498, 501, 504, 507, 510, 513, 516, 519, 522, 525 or528; and (iii) a VL CDR3 comprising the sequence shown in SEQ ID NO:195, 198, 201, 204, 207, 210, 213, 216, 219, 222, 225, 228, 231, 234,237, 240, 243, 246, 249, 252, 255, 258, 261, 264, 466, 469, 472, 475,478, 481, 484, 487, 490, 493, 496, 499, 502, 505, 508, 511, 514, 517,520, 523, 526 or 529.

In another aspect, provided is an isolated antibody which specificallybinds to CD70, wherein the antibody comprises: a VH region comprising aVH CDR1, VH CDR2, and VH CDR3 of the VH sequence shown in SEQ ID NO: 2,4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40,42, 44, 46, 48, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309,311, 313, 315, 317, 319, 321, 323, 325, 327, 329 or 331; or a VL regioncomprising VL CDR1, VL CDR2, and VL CDR3 of the VL sequence shown in SEQID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33,35, 37, 39, 41, 43, 45, 47, 288, 290, 292, 294, 296, 298, 300, 302, 304,306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328 or 330.

In some embodiments, provided is an antibody having any one of partiallight chain sequence as listed in Table 1 or any one of partial heavychain sequence as listed in Table 1. In Table 1, the underlinedsequences are CDR sequences according to Kabat and in bold according toChothia.

TABLE 1 mAb Light Chain Heavy Chain 31H1 DIVMTQNPLSSPVTLGQPASISC RSQVQLVQSGAEVKKPGSSVKVSCKA SQSLVHSDGNTYLS WLQQRPGQS SGGTFSSYGFSWVRQAPGQGLEW PRLLIY KISNRFS GVPDRFSGSGAG MGGIIPIFGSANYAQKFQGRVTITADTDFTLKISRVEAEDVGVYYC MQAT KSTSTVYMELISLRSEDTAVYYCAR QFPLT IGGGSKVEIKGGSSSPFAY WGQGTLVTVSS (SEQ ID NO: 1) (SEQ ID NO: 2) 63B2DIVMTQTPLSSPVTLGQPASISC RS QVQLVQSGAEVKKPGSSVKVSCKA SQSLVHSDGNTYLSWLQQRPGQS SGGTFS SYGFSWVRQAPGQGLEW PRLLIY KISNRFS GVPDRFSGSGAGMGGIIPIFGTANYAQKFQGRVTITAD TDFTLKISRVEAEDVGVYYC MQATKSTSTVFMELISLRSEYTAVYYCAR QFPLT IGGGSKVEIK GGSSSPFAY WGQGTLVTVSS(SEQ ID NO: 3) (SEQ ID NO: 4) 40E3 DIQMTQSPSSLSASVGDRVTITC RAQVQLQESGPGLVKPSETLSLTCTVS SQGISNYLA WFQQKPGKAPKSLIY GGSISSYYWNWIRQPPGKGLEWIG AASSLQS GVPSKFSGSGSGTDFTL YIYYSGSTNYNPSLKSRVTISVDTSKTISSLQPEDFATYYC QQYNSYPLT F NQFSLKLRSVTAADTAVYYCAR DIR GGGTKVEIK TWGQGTLVTVSS (SEQ ID NO: 5) (SEQ ID NO: 6) 42C3 DVVMTQSPLSLPVTLGQPASISC RSEVQLVESGGGLVQPGGSLRLSCAA SQSLVYSDENTYLN WFQQRPGQS SGFTFRNSWMSWVRQAPGKGLEW LRRLIY QVSNRDS GVPDRFSGSGS VANIKRDGSEKYYVDSVKGRFTISRGTDFTLKISRVEAEDVGVYFC MQG DNAKNSLYLQMNSLRAEDTAVYYC TYWPPT FGGGTKVEIK ARDQTGSFDY WGQGTLVTVSS (SEQ ID NO: 7) (SEQ ID NO: 8) 45F11EIVMTQSPATLSMSLGERATLSC RA QVQLRGSGPGLVKPSETLSLTCTVS SQSVSSSLAWYQQKPGQAPRLLIY DDSIS VYYWSWIRQPAGKGLEWIGR GASTRAT GIPARFGGSGSGTEFTLVYSSGNINYNPSLESRVTMSVDTSK TISSLQSEDFAVYYC QQYINWPH FGSRFSLNLSSVTAADTAVYYCAR GLD GGTKVEIK AFDI WGQGTMVTVSS (SEQ ID NO: 9)(SEQ ID NO: 10) 64F9 DIQMTQSPSSLSASVGDRVTITCQA EVQLLESGGGLVQPGESLRLSCEVSSQDISNYLNWYQQKPGKAPKILIYG GFTFT SYAMSWVRQVPGKGLEWVSASNLETGVPSRFSGSGSGTDFTFAI IISGVAFTTYYADSVKGRFTISRDHSSSLQPEDVATYYCQQYDNFPITFG KNTLYLQMNGLRAEDTAVYYCVK V QGTRLEIK DGEVYWGQGTLVTVSS (SEQ ID NO: 11) (SEQ ID NO: 12) 72C2EIVMTQSPDTLSVSPGERAILSCRA QVQLVQSGAEVKKPGSSVKVSCEASQSVSSNLAWYQQKPGQAPRLLIY SGGTFI TYAISWVRQAPGQGLEWMSASTRASGIPARFSGSGSGTEFTLS GGIIPFFGTANYAQKFQGRVTITADKISSLQSEDFAVYYCQQYDNWPPLT STSTASMELRSLRSEDTAMYYCAQ FGGGTKVEIK WELFFFDFWGQGTPVTVSS (SEQ ID NO: 13) (SEQ ID NO: 14) 2F10EIVLTQSPGTLSLSPGERATLSCRA AVQLVESGGGLVQPGGSLRLSCAASQSVSSSYLAWYQQQPGQAPRLLI SGFTFT YYSMNWVRQAPGKGLEWYGASSRATGIPDRFSGSGSGTDFT VSHISIRSSTIYFADSAKGRFTISRDNLTISRLEPEDFAIYYCQQYGSSPLTF AKNSLYLQMNSLRDEDTAVYYCAR GGGTKVEIKGSGWYGDYFDYWGQGTLVTVSS (SEQ ID NO: 15) (SEQ ID NO: 16) 4F11DIQMTQSPSAMSASVGDRVTITCR QVTLKESGPVLVKPTETLTLTCTVSASQDISNYLAWFQQKPGKVPKRLI GFSLS NARMGVTWIRQPPGKALEWYAASSLQSGVPSRFSGSGSGTEFT LAHIFSNDEKSYSTSLKSRLTISKDTLTISSLLPEDFATYYCLQLNSFPFTF SKTQVVLTMTNMDPVDTATYYCAR I GGGTKVEINRDYYDISSYYDY WGQGTLVSVSS (SEQ ID NO: 17) (SEQ ID NO: 18) 10H10DIQMTQSPSSVSASVGDRVTITC RA EVQLVESGGGLVQPGGSLRLSCAV SQGISSWLAWYQQKPGKAPKVLIY SGFTFS NHNIHWVRQAPGKGLEWIS AASSLQS GVPSRFSGSGSGTDFTLYISRSSSTIYYADSVKGRFTISRDNA TISSLQPEDFATYYC QQAFSFPFT FKNSLYLQMNSLRDEDTAVYYCAR D GPGTKVDIK HAQWYGMDV WGQGTTVTVSS(SEQ ID NO: 19) (SEQ ID NO: 20) 17G6 DIVMTQSPDSLAVSLGERATINC KSEVQLVESGGGLVQPGGSLRLSCVA SQSVLYSYNNKNYVA WYQQKPGQ SGFTFSSYWMSWVRQAPGKGLEW PPNLLIF WASTRES GVPDRFSGSG VASIKQDGSEKYYVDSVKGRFTISRSGTDFTLTISSLQAEDVAVYYC QQY DNAKNSVYLQMNSLRAEDTGVYYC YSTLT FGGGTKVEIK AREGVNWGWRLYWHFDL WGRGTL (SEQ ID NO: 21) VTVSS (SEQ ID NO: 22) 65E11EIVLTQSPGTLSLSPGERVTLSC RA EVQVVESGGGLVQPGGSLRLSCAA SQSVSSSYLAWYQQKPGQAPRLLI SGFTFS SYSMNWVRQAPGKGLEW Y DASSRAT GIPDRFSGSGSGTDFTVSHSSISRGNIYFADSVKGRFTISRD LTISRLEPEDFAVYYC QQYGSSPLTNAKNSLYLQMNSLRDEDTAVYYCA FGGGTKVEIK R GSGWYGDYFDY WGQGTLVTVSS(SEQ ID NO: 23) (SEQ ID NO: 24) P02B10 ELQSVLTQPPSASGTPGQRVTISC SEVQLLESGGGLVQPGGSLRLSCAA GSSSNIGSNYVY WYQQLPGTAPKL SGFAFSNYAMSWVRQAPGKGLEW LIY RNNQRPS GVPDRFSGSKSGTS VSAIRGGGGSTYYADSVKGRFTISRASLAISGLRSEDEADYYC AAWDDS DNSKNTLYLQMNSLRAEDTAVYYCA LSGVV FGGGTKLTVL RDFISGTWYPDY WGQGTLVTVSS (SEQ ID NO: 25) (SEQ ID NO: 26) P07D03ELQSVLTQPPSASGTPGQRVTISC S EVQLVQSGAEVKKPGESLKISCKGS GSRSNIGSNYVYWYQQLPGTAPKL GYRFT SYWIGWVRQMPGKGLEWM LIY RNNQRPS GVPDRFSGSKSGTSGSIYPDDSDTRYSPSFQGQVTISAD ASLAISGLRSEDEADYYC ASWDGSKSISTAYLQWSSLKASDTAMYYCAS LSAVV FGTGTKLTVL STVDYPGYSYFDY WGQGTLVTVSS(SEQ ID NO: 27) (SEQ ID NO: 28) P08A02 ELQSVLTQPPSASGTPGQRVTISC SEVQLVQSGAEVKKPGESLKISCKGS GSSSNIGSNYVY WYQQLPGTAPKL GYTFTNYWIAWVRQMPGKGLEWM LIY RNNQRPS GVPDRFSGSKSGTS GIIYPDGSDTRYSPSFQGQVTISADKASLAISGLRSEDEADYYC ATWDDS SISTAYLQWSSLKASDTAMYYCAR D LGSPV FGTGTKLTVLITS W YYGEPAFDI WGQGTLVTVSS (SEQ ID NO: 29) (SEQ ID NO: 30) P08E02ELDIQMTQSPSSLSASVGDRVTITC EVQLVQSGAEVKKPGESLKISCKGS RASQSISRYLNWYQQKPGKAPKLLI GYSFT SSWIGWVRQMPGKGLEWM Y AASILQT GVPSRFSGSGSGTDFTGIIYPGDSDTRYSPSFQGQVTISADK LTISSLQPEDFATYYC QQSYSTTMSISTAYLQWSSLKASDTAMYYCAK G WT FGQGTKVEIK LSQAMTGFGFDY WGQGTLVTVSS(SEQ ID NO: 31) (SEQ ID NO: 32) P08F08 ELQSVLTQPPSASGTPGQRVTISC SEVQLVQSGAEVKKPGESLKISCKGS GSSSNIGSNYVN WYQQLPGTAPKL GYGFTSYWIGWVRQMPGKGLEWM LIY GDYQRPS GVPDRFSGSKSGTS GIIHPDDSDTKYSPSFQGQVTISADKASLAISGLRSEDEADYYC ATRDDSL SISTAYLQWSSLKASDTAMYYCAS S SGSVV FGTGTKLTVLYLRGLWGGYFDY WGQGTLVTVSS (SEQ ID NO: 33) (SEQ ID NO: 34) P08G02ELDIQMTQSPSSLSASVGDRVTITC EVQLVQSGAEVKKPGESLKISCKGS RASQSIYDYLHWYQQKPGKAPKLLI GYTFP SSWIGWVRQMPGKGLEWM Y DASNLQS GVPSRFSGSGSGTDFTGIIYPDTSHTRYSPSFQGQVTISADK LTISSLQPEDFATYYC QQSYTTPLFSISTAYLQWSSLKASDTAMYYCAR A T FGQGTKVEIK SYFDRGTGYSSWWMDV WGQGTLV(SEQ ID NO: 35) TVSS (SEQ ID NO: 36) P12B09 ELDIQMTQSPSSLSASVGDRVTITCEVQLLESGGGLVQPGGSLRLSCAA RASQYIGRYL NWYQQKRGKAPKLL SGFTFSQYSMSWVRQAPGKGLEW IH GATSLAS GVPSRFSGSGSGTDF VSAISGGGVSTYYADSVKGRFTISRTLTISSLQPEDFATYYC QQSYSTTS DNSKNTLYLQMNSLRAEDTAVYYCA PT FGQGTKVEIK SDISDSGGSH W YFDY WGQGTLVTV (SEQ ID NO: 37) SS (SEQ ID NO: 38) P12F02ELQSVLTQPPSASGTPGQRVTISC S EVQLLESGGGLVQPGGSLRLSCAA GSTSNIGRNYVYWYQQLPGTAPKL SGFTFS SYAMSWVRQAPGKGLEW LIY RTNQRPS GVPDRFSGSKSGTSVSTISGTGGTTYYADSVKGRFTISRD ASLAISGLRSEDEADYYC AAWDDSNSKNTLYLQMNSLRAEDTAVYYCAK LSGRV FGTGTKLTVL VRAGIDPTASDV WGQGTLVTVSS(SEQ ID NO: 39) (SEQ ID NO: 40) P12G07 ELQSVLTQPPSASGTPGQRVTISC SEVQLLESGGGLVQPGGSLRLSCAA GSSSNIGSNYVY WYQQLPGTAPKP SGFTFNNFAMSWVRQAPGKGLEW LIY MNNQRPS GVPDRFSGSKSGTS VSGISGSGDNTYYADSVKGRFTISRASLAISGLRSEDEADYYC AAWDDS DNSKNTLYLQMNSLRAEDTAVYYCA LSAVV FGTGTKLTVL KDRDIGLGWYSYYLDV WGQGTLVT (SEQ ID NO: 41) VSS (SEQ ID NO: 42) P13F04ELQSVLTQPPSASGTPGQRVTISC S QVQLVQSGAEVKKPGSSVKVSCKA GSNSNIGTNYVSWYQQLPGTAPKL SGGTFS SYAISWVRQAPGQGLEWM LIY RSSRRPS GVPDRFSGSKSGTSGEIIPIFGTASYAQKFQGRVTITADES ASLAISGLRSEDEADYYC AAWDGSTSTAYMELSSLRSEDTAVYYCAR AG LSGHWV FGTGTKLTVL WDDSWFDY WGQGTLVTVSS(SEQ ID NO: 43) (SEQ ID NO: 44) P15D02 ELDIQMTQSPSSLSASVGDRVTITCEVQLVQSGAEVKKPGESLKISCKGS RASQSIDTYLN WYQQKPGKAPKLLI GYSFASYWIGWVRQMPGKGLEWM Y SASSLHS GVPSRFSGSGSGTDFT GVIYPGTSETRYSPSFQGQVTISADLTISSLQPEDFATYYC QQSYSTTAW KSISTAYLQWSSLKASDTAMYYCAK T FGQGTKVEIKGLSASASGYSFQY WGQGTLVTVSS (SEQ ID NO: 45) (SEQ ID NO: 46) P16C05ELDIQMTQSPSSLSASVGDRVTITC EVQLVQSGAEVKKPGESLKISCKGS RASQSIGQSLNWYQQKPGKAPKLL GYSFT DYWIGWVRQMPGKGLEWM IY GASSLQS GVPSRFSGSGSGTDFGMISPGGSTTIYRPSFQGQVTISADK TLTISSLQPEDFATYYC QQSYSTPITSISTAYLQWSSLKASDTAMYYCAR E FGQGTKVEIK MYTGGYGGSWYFDY WGQGTLVTV(SEQ ID NO: 47) SS(SEQ ID NO: 48) 10A1 DIQMTQSPSTLSASVGDRVTITC RAQVQLQESGPGLVKPSETLSLTCTVS SQSISTWLA WYQQKPGKAPKVLIY GGSISYYYWTWIRQPPGKGLEWIG KASSLES GVPSRFSGSGSGTEFILTHIYYSGSTNYNPSLKSRVTISIDTSK INSLQPDDFASYYC QQYKSYSHT FNLFSLKLSSVTAADTAVYYCAR AEG GQGTKLEIK SIDAFDF WGQGTMVTVSS(SEQ ID NO: 288) (SEQ ID NO: 289) 10E2 DIQMTQSPSTLSASVGDRVTITC RAEVQLVESGGGLIQPGGSLRLSCAAS SQSISSWLA WYQQKPGKAPKVLIY GFTVSSNYMTWVRQAPGKGLEWV KASSLES GVPSRFSGSGSGTEFTL SVIYSGGSTYYADSVKGRFTISRDNTINSLQPDDFATYYC QQYKSFSLT F SKNTLYLQMNSLRAEDTAVYYCAR N GQGTKLEIK WGDYWGQGTLVTVSS (SEQ ID NO: 290) (SEQ ID NO: 291) 11A1DIQMTQSPSTLSASVGDRVTITC RA QVQLQESGPGLVKPSGTLSLTCTVS SQSISSWLAWYQQKPGKAPKVLIY GGSID YYFWNWFRQSPVKGLEWIG KASTLES GVPSRFSGSGSGTEFTLHVYDIGNTKYNPSLKSRVTISIDTSE TISSLQPDDFATYYC QQYNSYSYT FNQFSLKLNSVTAADTAVYYCAR GEG GHGTKLEIK AIDAFDI WGQGTMVTVSS(SEQ ID NO: 292) (SEQ ID NO: 293) 11C1 DIQMTQSPSILSASVGDRVTITC RAQVQLQESGPGLVKPSETLSLNCTVS SQSVSSWLA WYQQKPGKAPKVLIY GGSISYYYWTWIRQPPGKGLEWIG KASSLES GVPSRFSGTGSGTEFTL HVIYSGTTNYNPSLKSRVTISVDTSKTISSLQSDDFATYYC QQYNTYSHT F NQFSLKLNSVTAADTAVYYCVR AEG GQGTKLEIK SIDAFDLWGQGTMVTVSS (SEQ ID NO: 294) (SEQ ID NO: 295) 11D1AIQMTQSPSSLSASVGDRVTITC RA QVQLVESGGGVVQPGRSLRLSCVA SQGIRNDLGWYQQKPGKAPKLLIY SGFTFS DYGIHWVRQAPGMGQEW AASSLQS GVPSRFSGSGSGTDFTLVAVIWYDGSiKKYSDSVKGRFIISRD TISSLQPEDFATYYC LQDYNYPFT FNSENTVYLQMNSLRGEDTAIYYCAR GPGTKVDIK DEVGtfGAFDF WGQGTKVTVSS(SEQ ID NO: 296) (SEQ ID NO: 297) 11E1 DIQMTQSPSSLSASVGDSITITC RAQVQLQESGPGLVKPLQTLSLTCTVS SQDIDNYLA WYQQKTGKVPKVLIY GGSISSdgYYWSWIRQNPGKGLEWI AASALQS GVPSRFSGSGSGTDFTL GYMYYSGSTYYNPSLKSRVTISVDTTISSLQPEDVATYYC QNYNSGPRT F SKNQFSLKLRSVTAADTAVYYCTR D GQGTKVEIK FG WYFDL WGRGTLVTVSS (SEQ ID NO: 298) (SEQ ID NO: 299) 12A2DIQMTQSPSSLSASVGDRVTITC RA QVQLQESGPGLVKPSQSLSLTCSVS SQDISNYLTWYQQKPGRVPEVLIY GGSVS SdgYYWSWIRQHPGKGLEW AASALQS GVPSRFSGSGSGTDFTLIGYIYYRRITDYNPSLKSRVNISLDTS TISSLQPEDVATYYC QNYNSAPRT FKNQFSLKLSSVTAADTAVYYCAR DF GQGTKVEIK GWYFDL WGRGTLVAVSS (SEQ ID NO: 300)(SEQ ID NO: 301) 12C4 DIVMTQSPLSLPVTPGEPASISC RSQVQLVQSGAEVKKPGASVKVSCKA SQSLLHSNGYNYLD WYLQKPGQS SGYTFTGYYLHWVRQAPGQGLEW PQVLIL LGSNRAS GVPDRVSASGS MGWINpNSGGTNYAQKFQGRVTMTGTDFTLKISRMQAEDVGIYYC MQTL RDTSITTAYMELSRLRIDDTAVYYCA QTPFTF GQGTKLEIK RDRGVtmivDGMDD WGQGTTVTVS (SEQ ID NO: 302) S (SEQ ID NO: 303) 12C5DIQLTQSPSFLSASVGDRVIITC RAS EVELVESGGGMVQPGRSLRLSCAA QGINSHLAWYQQKPGKAPKLLIY Y SGFTFS DYGMHWVRQAPGMGLEW ASTLPS GVPSRFSGSGSGTEFTLTVTVIW YDGSnKYYADSVKGRFTISR VTSLQPEDFATYYC QQLNHYPIT FGDNSKNTVFLQMNSLRAEDTAVYYC QGTRLDIN AR DEVGfvGAFDI WGQGTMVTVSS(SEQ ID NO: 304) (SEQ ID NO: 305) 12D3 DIQMTQSPSSLSASVGDRVTITC RAQVQLQESGPGLVKPSQTLSLTCTVS SQGISNYLA WYQQKPGKVPKLLIY GGSIS Sd gYYWSWIRQHPGKGLEWI AASTLHS GVPSRFSGSGSGTDFTL GYMYYSGITYHNPSLKSRVTISVDTSTISSLQPEDVATYYC QKYNSAPRT F KNQFSLRLSSVTAADTAVYYCAR DF GQGTKVEIK GWYFDLWGRGTLVTVSS (SEQ ID NO: 306) (SEQ ID NO: 307) 12D6DIQMTQSPSSLSASVGDRVTITC RA QVQLQESGPGLVKPSQTLSLTCTVS SQDISNYLAWYQQKPGKVPKLLIYA GGSIS SdaYYWSWIRQHPGKGLEWI ASTLHS GVPSRFSGSGSGTDFTLTIGYMYYSGITYYNPSLKSRVTISVDTS SSLQPDDFAAYYC QKYNSAPRT FGKNQFSLKLSSVTAADTAVYYCAR DF QGTKVEIK GWYFDL WGRGTLVTVSS (SEQ ID NO: 308)(SEQ ID NO: 309) 12D7 DIQLTQSPSFLSASVGDRVSITC RAQVQLVESGGGVVQPGRSLRLSCVA SQDISSFLA WYQQKPGKAPVLLIYV SGFTFSDYGIHWVRQAPGMGQEW ASTLQS GVPSRFSGSGSGTEFTLT VAVIWYDGS iKKYSDSVKGRFIISRDVSSLQPEDFATYYC QQLHVYPIT FG NSENTVYLQMNSLRGEDTAIYYCAR QGTRLEIRDEVGtfGAFDF WGQGTKVIVSS (SEQ ID NO: 310) (SEQ ID NO: 311) 12F5DIVMTQTPLSLPVTPGEPASISC RS EVQLVESGGGLVKPGGSLRLSCAA SQSLLDSDDGNtYLDWYLQKPGQS SGFTFS NAWMSWVRQAPGKGLEW PQLLIY TLSYRAS GVPDRFSGSGSVGRIKsktGGGTTDYAAPVKGRFTIS GTDFTLKISRVEAEDVGVYYC MQRIRDDSKNTLYLQMNSLKTEDTAVYYC EFPFT FGPGTKVDIK TS LIVGaiSLFDY WGQGTLVTVSS(SEQ ID NO: 312) (SEQ ID NO: 313) 12H4 DIQMTQSPSALSASVGDRVAITC RAQVQLRESGPGLVKPSETLSLTCTIS SQTISTWLA WYQQKPGKAPKVLIY GGSISYYFWTWIRQPPGRGLEWIG KASNLES GVPSRFSGSGSGTEFTL QIYYSGNTNSNPSLKSRVTISIDTSKTINSLQPDDFATYYC QQYQTFSHT F NQFSLKLTSVTVADTAVYYCVR AEG GQGTKLEIK SIDAFDIWGQGTMVAVSS (SEQ ID NO: 314) (SEQ ID NO: 315) 8C8DMQMTQSPSSLSASVGDRVTLTC R EVQLVESGGGLVKPGGSLRLSCVA ASQGISNYLAWFQLKPGKVPKLLIY SGFTFS SYSMNWVRQFPGKGLEW AASTLQS GVPSRFSGSGSGTDFALVSSIStSSNYIHYADSLQGRFTISRDN TISSLQPEDVATYYC QKYNSAPLT FAKNSLYLQMSSLRVEDTAVYYCVR D GGGTKVEIK KGTtltnWYFDL WGRGTLVTVSS(SEQ ID NO: 316) (SEQ ID NO: 317) 8F7 DIVMTQSPLSLPVTPGEPASISC RSQVQLVESGGGVVQPGRSLRLSCGA SQTLVHSNGYNYLN WYLQKPGQS SGFTFSSYGMHWVRQAPGKGLEW PQLLIY LGSNRAS GVPDRFSGSGS VAVIW YDGSnKYYADSLKGRFTISRGSDFTLKISRMEAEDVGVYYC MQA DNSKNTLYLQMNSLRAEDTAVYYCA IQTPYT FGQGTNVEIK RDGYS g ssDAFDI WGQGTMVTVSS (SEQ ID NO: 318) (SEQ ID NO: 319) 8F8DIQMTQSPSTLSASVGDRVTITC RA QVQLQESGPGLVQPSETLSLTCTVS SQSISSWLAWYQQKPGKAPKVLIY GGSIS YYYWSWIRQPPGKGLEWIG KASNLES GVPSRFSGSGSGTEFTLNINYMGNTIYNPSLKSRVTISVDTSK TISSLQPDDFATYYC QQYNSYSCT FDQFSLKLTSVSAADTAVYYCVR AEG GQGTKLEIK SIDAFDF WGQGTLVAVSL(SEQ ID NO: 320) (SEQ ID NO: 321) 9D8 DIQMTQSPSSLSASVGDRIIFTC QAQVQLVQSGAEVTKPGASVKVSCKA SQDINNYLH WYQQKPGKAPKLLIY SGYIFTGYYIYWVRQAPGQGLEWM DASDWET GVPSRFSGSGSGTDFT GWINpSSGGTNYAQKFQGRVTMARFTISSLQPEDIATYYC QQYDHLPIT F DTSISTAYMELSSLRSDDTAVYYCA GQGTRVEIK RDRKReyyynFGMDV WGQGTTVTV (SEQ ID NO: 322) ST (SEQ ID NO: 323) 9E10DIQMTQSPSSLSASVGDRVILTC QA QVQLVQSGAEVTKPGASVKVSCKA SQDISNYLHWYQQKPGKAPKLLIYD SGYTFT SHYIYWVRQAPGQGLEWM ASDLET GVPSRFSGSGSGADFTFTIGWINpNSGGTNYAQKFQDRVTMAR SNLQPEDFATYYC QQYDHLPIT FGDTSISTAYMELSRLRSDDTAVYYCA QGTRLEIK K DRKReyyynFGMDV WGQGTTVTV(SEQ ID NO: 324) SA (SEQ ID NO: 325) 9E5 DIQMTQSPSSLSASVGDRVILTC QAQVQLVQFGVEVRKPGASVKVSCKV SQDISNYLH WYQQKPGKAPKLLIYD SGFTFTSHYIYWVRQAPGQGLEWM ASDLET GVPSRFSGSGSGADFTFTI GWINpNSGGTKYAQKFQDRVTMARSNLQPEDFATYYC QQYDHLPIT FG DTSISTAYMELSRLRSDDTSVYYCV QGTRLEIK KDRKReyyynFGMDV WGQGTTVTV (SEQ ID NO: 326) SS (SEQ ID NO: 327) 9F4DIQMTQSPSSLSASVGDRVTITC QA EVQMLESGGGLIQPGGSLRLSCKTS SQDISNYLNWYQQKPGKAPKLLIY D GFTLS IYAIHWVRQAPGRGLEWVSS ASNLET GVPSRFSGSGSGTDFTFTIFGgRGSSTYFADSVKGRFTISRDAS SSLQPEDIATYYC QQYDNLPYT FGENSLYLHMNSLRAEDTAVYYCAK EK QGTKLEIK DWgRGFDY WGQGTLVTVSS(SEQ ID NO: 328) (SEQ ID NO: 329) 9F8 DIVMTQSPLSLPVTPGEPASISC RSEVQLVESGGGLVKPGGSLRLSCAA SQSLLYSNGYNYLD WYLQKPGQS SGFTFSNYSMNWVRQAPGKGLEW PQLLIF LNSNRAS GVPDRFSGSGS VSSISsSTIYIYYADSVKGRFTISRDNGTDFTLKISRVEAEDVGVYFC MQA AKKSLYLQMNSLRAEDTAVYYCAR D LQTPLT FGGGTKVEIKIGWevftLGFDY WGQGTQVTVSS (SEQ ID NO: 330) (SEQ ID NO: 331)

Also provided herein are CDR portions of antigen binding domains ofantibodies to CD70 (including Chothia, Kabat CDRs, and CDR contactregions). Determination of CDR regions is well within the skill of theart. It is understood that in some embodiments, CDRs can be acombination of the Kabat and Chothia CDR (also termed “combined CRs” or“extended CDRs”). In some embodiments, the CDRs are the Kabat CDRs. Inother embodiments, the CDRs are the Chothia CDRs. In other words, inembodiments with more than one CDR, the CDRs may be any of Kabat,Chothia, combination CDRs, or combinations thereof. Table 2 providesexamples of CDR sequences provided herein.

TABLE 2 Heavy Chain mAb CDRH1 CDRH2 CDRH3 31H1 SYGFS (SEQ ID NO: 49)GIIPIFGSANYAQK GGSSSPFAY (Kabat); FQG (SEQ ID NO: (SEQ ID NO: 54)GGTFSSY (SEQ ID NO: 52) (Kabat); 50) (Chothia); IPIFGS (SEQ IDGGTFSSYGFS (SEQ ID NO: 53) (Chothia) NO: 51) (Extended) 63B2SYGFS (SEQ ID NO: 55) GIIPIFGTANYAQK GGSSSPFAY (Kabat); FQG (SEQ ID NO:(SEQ ID NO: 60) GGTFSSY (SEQ ID NO: 58) (Kabat); 56) (Chothia)IPIFGT (SEQ ID GGTFSSYGFS NO: 59) (Chothia) (Extended) (SEQ ID NO: 57)40E3 SYYWN (SEQ ID NO: 61) YIYYSGSTNYNPS DIRTW (SEQ ID (Kabat);LKS (SEQ ID NO: NO: 66) GGSISSY (SEQ ID NO: 64) (Kabat); 62) (Chothia);YYSGS (SEQ ID GGSISSYYWN (SEQ ID NO: 65) (Chothia) NO: 63) (Extended)42C3 NSVVMS (SEQ ID NO: NIKRDGSEKYYV DQTGSFDY (SEQ 67) (Kabat);DSVKG (SEQ ID ID NO: 72) GFTFRNS (SEQ ID NO: NO: 70) (Kabat);68) (Chothia); KRDGSE (SEQ ID GFTFRNSWMS (SEQ ID NO: 71) (Chothia)NO: 69) (Extended) 45F11 VYYWS (SEQ ID NO: 73) VYSSGNINYNPSLGLDAFDI (SEQ ID (Kabat); ES (SEQ ID NO: NO: 78) DDSISVY (SEQ ID NO:76) (Kabat); 74) (Chothia); YSSGN (SEQ ID DDSISVYYWS (SEQ IDNO: 77) (Chothia) NO: 75) (Extended) 64F9 SYAMS (SEQ ID NO: 79)RVYSSGNINYNP GLDAFDI (SEQ ID (Kabat); SLES (SEQ ID NO: 84)GFTFTSY (SEQ ID NO: NO: 82) (Kabat); 80) (Chothia); YSSGN (SEQ IDGFTFTSYAMS (SEQ ID NO: 83) (Chothia) NO: 81) (Extended) 72C2TYAIS (SEQ ID NO: 85) GIIPFFGTANYAQ WELFFFDF (SEQ (Kabat); KFQG (SEQ IDID NO: 90) GGTFITY (SEQ ID NO: NO: 88) (Kabat); 86) (Chothia);IPFFGT (SEQ ID GGTFITYAIS (SEQ ID NO: 89) (Chothia) NO: 87) (Extended)2F10 YYSMN (SEQ ID NO: 91) HISIRSSTIYFADS GSGWYGDYFDY (Kabat);AKG (SEQ ID NO: (SEQ ID NO: 96) GFTFTYY (SEQ ID NO: 94) (Kabat);92) (Chothia); SIRSST (SEQ ID GFTFTYYSMN (SEQ ID NO: 95) (Chothia)NO: 93) (Extended) 4F11 NARMGVT (SEQ ID NO: HIFSNDEKSYSTS IRDYYDISSYYDY97) (Kabat); LKS (SEQ ID NO: (SEQ ID NO: 102) GFSLSNARM (SEQ ID100) (Kabat); NO: 98) (Chothia); FSNDE (SEQ ID GFSLSNARMGVT (SEQNO: 101) (Chothia) ID NO: 99) (Extended) 10H10 NHNIH (SEQ ID NO: 103)YISRSSSTIYYAD DHAQWYGMDV (Kabat); SVKG (SEQ ID (SEQ ID NO: 108)GFTFSNH (SEQ ID NO: NO: 106) (Kabat); 104) (Chothia); SRSSST (SEQ IDGFTFSNHNIH (SEQ ID NO: 107) (Chothia) NO: 105) (Extended) 17G6SYWMS (SEQ ID NO: SIKQDGSEKYYV EGVNWGWRLYW 109) (Kabat); DSVKG (SEQ IDHFDL (SEQ ID GFTFSSY (SEQ ID NO: NO: 112) (Kabat); NO: 114)110) (Chothia); KQDGSE (SEQ ID GFTFSSYWMS (SEQ ID NO: 113) (Chothia)NO: 111) (Extended) 65E11 SYSMN (SEQ ID NO: HSSISRGNIYFAD GSGWYGDYFDY115) (Kabat); SVKG (SEQ ID (SEQ ID NO: 120) GFTFSSY (SEQ ID NO:NO: 118) (Kabat); 116) (Chothia); SISRGN (SEQ ID GFTFSSYSMN (SEQ IDNO: 119) (Chothia) NO: 117) (Extended) P02B10 NYAMS (SEQ ID NO:AIRGGGGSTYYA DFISGTWYPDY 121) (Kabat); DSVKG (SEQ ID (SEQ ID NO: 126)GFAFSNY (SEQ ID NO: NO: 124) (Kabat); 122) (Chothia); RGGGGS (SEQ IDGFAFSNYAMS (SEQ ID NO: 125) (Chothia) NO: 123) (Extended) P07D03SYWIG (SEQ ID NO: SIYPDDSDTRYSP STVDYPGYSYFD 127) (Kabat); SFQG (SEQ IDY (SEQ ID NO: GYRFTSY (SEQ ID NO: NO: 130) (Kabat); 132) 128) (Chothia);YPDDSD (SEQ ID GYRFTSYWIG (SEQ ID NO: 131) (Chothia) NO: 129) (Extended)P08A02 NYWIA (SEQ ID NO: IIYPDGSDTRYSP DITSWYYGEPAF 133) (Kabat);SFQG (SEQ ID DI GYTFTNY (SEQ ID NO: NO: 136) (Kabat); (SEQ ID NO: 138)134) (Chothia); YPDGSD (SEQ ID GYTFTNYWIA (SEQ ID NO: 137) (Chothia)NO: 135) (Extended) P08E02 SSWIG (SEQ ID NO: IIYPGDSDTRYSP GLSQAMTGFGFD139) (Kabat); SFQG (SEQ ID Y (SEQ ID NO: GYSFTSS (SEQ ID NO:NO: 142) (Kabat); 144) 140) (Chothia); YPGDSD (SEQ ID GYSFTSSWIG (SEQ IDNO: 143) (Chothia) NO: 141) (Extended) P08F08 SYWIG (SEQ ID NO:IIHPDDSDTKYSP SYLRGLWGGYF 145) (Kabat); SFQG (SEQ ID DY (SEQ ID NO:GYGFTSY (SEQ ID NO: NO: 148) (Kabat); 150) 146) (Chothia);HPDDSD (SEQ ID GYGFTSYWIG (SEQ ID NO: 149) (Chothia) NO: 147) (Extended)P08G02 SSWIG (SEQ ID NO: IIYPDTSHTRYSP ASYFDRGTGYSS 151) (Kabat);SFQ (SEQ ID NO: WWMDV (SEQ ID GYTFPSS (SEQ ID NO: 154) (Kabat); NO: 156)152) (Chothia); YPDTSH (SEQ ID GYTFPSSWIG (SEQ ID NO: 155) (Chothia)NO: 153) (Extended) P12809 QYSMS (SEQ ID NO: AISGGGVSTYYA DISDSGGSHWYF157) (Kabat); DSVKG (SEQ ID DY (SEQ ID NO: GFTFSQY (SEQ ID NO:NO: 160) (Kabat); 162) 158) (Chothia); SGGGVS (SEQ ID GFTFSQYSMS (SEQ IDNO: 161) (Chothia) NO: 159) (Extended) P12F02 SYAMS (SEQ ID NO:TISGTGGTTYYAD VRAGIDPTASDV 163) (Kabat); SVKG (SEQ ID (SEQ ID NO: 168)GFTFSSY (SEQ ID NO: NO: 166) (Kabat); 164) (Chothia); SGTGGT (SEQ IDGFTFSSYAMS (SEQ ID NO: 167) (Chothia) NO: 165) (Extended) P12G07NFAMS (SEQ ID NO: GISGSGDNTYYA DRDIGLGWYSYY 169) (Kabat); DSVKG (SEQ IDLDV (SEQ ID NO: GFTFNNF (SEQ ID NO: NO: 172) (Kabat); 174)170) (Chothia); SGSGDN (SEQ ID GFTFNNFAMS (SEQ ID NO: 173) (Chothia)NO: 171) (Extended) P13F04 SYAIS (SEQ ID NO: 175) EIIPIFGTASYAQKAGWDDSWFDY (Kabat); FQG (SEQ ID NO: (SEQ ID NO: 180) GGTFSSY (SEQ ID NO:178) (Kabat); 176) (Chothia); IPIFGT (SEQ ID GGTFSSYAIS (SEQ IDNO: 179) (Chothia) NO: 177) (Extended) P15D02 SYWIG (SEQ ID NO:VIYPGTSETRYSP GLSASASGYSFQ 181) (Kabat); SFQG (SEQ ID Y (SEQ ID NO:GYSFASY (SEQ ID NO: NO: 184) (Kabat); 186) 182) (Chothia);YPGTSE (SEQ ID GYSFASYWIG (SEQ ID NO: 185) (Chothia) NO: 183) (Extended)P16C05 DYWIG (SEQ ID NO: MISPGGSTTIYRP MYTGGYGGSWY 187) (Kabat);SFQG (SEQ ID FDY (SEQ ID NO: GYSFTDY (SEQ ID NO: NO: 190) (Kabat); 192)188) (Chothia); SPGGST (SEQ ID GYSFTDYWIG (SEQ ID NO: 191) (Chothia)NO: 189) (Extended) 10A1 YYYWT (SEQ ID NO: HIYYSGSTNYNPS AEGSIDAFDF332) (Kabat); LKS (SEQ ID NO: (SEQ ID NO: 337) GGSISYY (SEQ ID NO:335) (Kabat); 333) (Chothia); YYSGS (SEQ ID GGSISYYYWT (SEQ IDNO: 336) (Chothia) NO: 334) (Extended) 10E2 SNYMT (SEQ ID NO:VIYSGGSTYYADS NWGDYW (SEQ 338) (Kabat); VKG (SEQ ID NO: ID NO: 343)GFTVSSN (SEQ ID NO: 341) (Kabat); 339) (Chothia); YSGGS (SEQ IDGFTVSSNYMT (SEQ ID NO: 342) (Chothia) NO: 340) (Extended) 11A1YYFWN (SEQ ID NO: HVYDIGNTKYNP GEGAIDAFDI 344) (Kabat); SLKS (SEQ ID(SEQ ID NO: 349) GGSIDYY (SEQ ID NO: NO: 347) (Kabat); 345) (Chothia);YDIGN (SEQ ID GGSIDYYFWN (SEQ ID NO: 348) (Chothia) NO: 346) (Extended)11C1 YYYWT (SEQ ID NO: HVIYSGTTNYNPS AEGSIDAFDL 350) (Kabat);LKS (SEQ ID NO: (SEQ ID NO: 355) GGSISYY (SEQ ID NO: 353) (Kabat);351) (Chothia); IYSGT (SEQ ID GGSISYYYWT (SEQ ID NO: 354) (Chothia)NO: 352) (Extended) 11D1 DYGIH (SEQ ID NO: 356) VIWYDGSiKKYSDDEVGtfGAFDF (Kabat); SVKG (SEQ ID (SEQ ID NO: 361) GFTFSDY (SEQ ID NO:NO: 359) (Kabat); 357) (Chothia); WYDGSi (SEQ ID GFTFSDYGIH (SEQ IDNO: 360) (Chothia) NO: 358) (Extended) 11E1 SdgYYWS (SEQ ID NO:YMYYSGSTYYNP DFGWYFDL (SEQ 362) (Kabat); SLKS (SEQ ID ID NO: 367)GGSISSdgY (SEQ ID NO: 365) (Kabat); NO: 363) (Chothia); YYSGS (SEQ IDGGSISSdgYYWS (SEQ NO: 366) (Chothia) ID NO: 364) (Extended) 12A2SdgYYWS (SEQ ID NO: YIYYRRITDYNPS DFGWYFDL (SEQ 368) (Kabat);LKS (SEQ ID NO: ID NO: 373) GGSVSSdgY (SEQ ID 371) (Kabat);NO: 369) (Chothia); YYRRI (SEQ ID GGSVSSdgYYWS (SEQ NO: 372) (Chothia)ID NO: 370) (Extended) 12C4 GYYLH (SEQ ID NO: WINpNSGGTNYA DRGVtmivDGMD374) (Kabat); QKFQG (SEQ ID D (SEQ ID NO: GYTFTGY (SEQ ID NO:NO: 377) (Kabat); 379) 375) (Chothia); NpNSGG (SEQ ID GYTFTGYYLH (SEQ IDNO: 378) (Chothia) NO: 376) (Extended) 12C5 DYGMH (SEQ ID NO:VIWYDGSnKYYA DEVGfvGAFDI 380) (Kabat); DSVKG (SEQ ID (SEQ ID NO: 385)GFTFSDY (SEQ ID NO: NO: 383) (Kabat); 381) (Chothia); WYDGSn (SEQ IDGFTFSDYGMH (SEQ ID NO: 384) (Chothia) NO: 382) (Extended) 12D3SdgYYWS (SEQ ID NO: YMYYSGITYHNP DFGWYFDL 386) (Kabat); SLKS (SEQ ID(SEQ ID NO: 391) GGSISSdgY (SEQ ID NO: 389) (Kabat); NO: 387) (Chothia);YYSGI (SEQ ID GGSISSdgYYWS (SEQ NO: 390) (Chothia)ID NO: 388) (Extended) 12D6 SdaYYWS (SEQ ID NO: YMYYSGITYYNPDFGWYFDL (SEQ 392) (Kabat); SLKS (SEQ ID ID NO: 397) GGSISSdaY (SEQ IDNO: 395) (Kabat); NO: 393) (Chothia); YYSGI (SEQ ID GGSISSdaYYWS (SEQNO: 396) (Chothia) ID NO: 394) (Extended) 12D7 DYGIH (SEQ ID NO: 398)VIWYDGSIKKYSD DEVGtfGAFDF (Kabat); SVKG (SEQ ID (SEQ ID NO: 403)GFTFSDY (SEQ ID NO: NO: 401) (Kabat); 399) (Chothia); WYDGSi (SEQ IDGFTFSDYGIH (SEQ ID NO: 402) (Chothia) NO: 400) (Extended) 12F5NAWMS (SEQ ID NO: RIKsktGGGTTDY LIVGaiSLFDY 404) (Kabat); AAPVKG (SEQ ID(SEQ ID NO: 409) GFTFSNA (SEQ ID NO: NO: 407) (Kabat); 405) (Chothia);KsktGGGT (SEQ GFTFSNAWMS (SEQ ID ID NO: 408) NO: 406) (Extended)(Chothia) 12H4 YYFWT (SEQ ID NO: QIYYSGNTNSNP AEGSIDAFDI 410) (Kabat);SLKS (SEQ ID (SEQ ID NO: 415) GGSISYY (SEQ ID NO: NO: 413) (Kabat);411) (Chothia); YYSGN (SEQ ID GGSISYYFWT (SEQ ID NO: 414) (Chothia)NO: 412) (Extended) 8C8 SYSMN (SEQ ID NO: SIStSSNYIHYADS DKGTtltnWYFDL416) (Kabat); LQG (SEQ ID NO: (SEQ ID NO: 421) GFTFSSY (SEQ ID NO:419) (Kabat); 417) (Chothia); StSSNY (SEQ ID GFTFSSYSMN (SEQ IDNO: 420) (Chothia) NO: 418) (Extended) 8F7 SYGMH (SEQ ID NO:VIWYDGSnKYYA DGYSgssDAFDI 422) (Kabat); DSLKG (SEQ ID (SEQ ID NO: 427)GFTFSSY (SEQ ID NO: NO: 425) (Kabat); 423) (Chothia); WYDGSn (SEQ IDGFTFSSYGMH (SEQ ID NO: 426) (Chothia) NO: 424) (Extended) 8F8YYYWS (SEQ ID NO: NINYMGNTIYNPS AEGSIDAFDF 428) (Kabat); LKS (SEQ ID NO:(SEQ ID NO: 433) GGSISYY (SEQ ID NO: 431) (Kabat); 429) (Chothia);NYMGN (SEQ ID GGSISYYYWS (SEQ ID NO: 432) (Chothia) NO: 430) (Extended)9D8 GYYIY (SEQ ID NO: 434) WINpSSGGTNYA DRKReyyynFGMD (Kabat);QKFQG (SEQ ID V (SEQ ID NO: GYIFTGY (SEQ ID NO: NO: 437) (Kabat); 439)435) (Chothia); NpSSGG (SEQ ID GYIFTGYYIY (SEQ ID NO: 438) (Chothia)NO: 436) (Extended) 9E10 SHYIY (SEQ ID NO: 440) WINpNSGGTNYADRKReyyynFGMD (Kabat); QKFQD (SEQ ID V (SEQ ID NO: GYTFTSH (SEQ ID NO:NO: 443) (Kabat); 4454) 441) (Chothia); NpNSGG (SEQ IDGYTFTSHYIY (SEQ ID NO: 444) (Chothia) NO: 442) (Extended) 9E5SHYIY (SEQ ID NO: 446) WINpNSGGTKYA DRKReyyynFGMD (Kabat); QKFQD (SEQ IDV (SEQ ID NO: GFTFTSH (SEQ ID NO: NO: 449) (Kabat); 451) 447) (Chothia);NpNSGG (SEQ ID GFTFTSHYIY (SEQ ID NO: 450) (Chothia) NO: 448) (Extended)9F4 IYAIH (SEQ ID NO: 452) SFGgRGSSTYFA EKDWgRGFDY (Kabat);DSVKG (SEQ ID (SEQ ID NO: 457) GFTLSIY (SEQ ID NO: NO: 455) (Kabat);453) (Chothia); GgRGSS (SEQ ID GFTLSIYAIH (SEQ ID NO: 456) (Chothia)NO: 454) (Extended) 9F8 NYSMN (SEQ ID NO: SISsSTIYIYYADS DIGWevftLGFDY458) (Kabat); VKG (SEQ ID NO: (SEQ ID NO: 463) GFTFSNY (SEQ ID NO:461) (Kabat); 459) (Chothia); SsSTIY (SEQ ID GFTFSNYSMN (SEQ IDNO: 462) (Chothia) NO: 460) (Extended) Light Chain mAb CDRL1 CDRL2 CDRL331H1 RSSQSLVHSDGNTYLS KISNRFS (SEQ MQATQFPLT (SEQ ID NO: 193);ID NO: 194) (SEQ ID NO: 195) 63B2 RSSQSLVHSDGNTYLS KISNRFS (SEQMQATQFPLT (SEQ ID NO: 196); ID NO: 197) (SEQ ID NO: 198) 40E3RASQGISNYLA (SEQ ID AASSLQS (SEQ QQYNSYPLT NO: 199); ID NO: 200)(SEQ ID NO: 201) 42C3 RSSQSLVYSDENTYLN QVSNRDS (SEQ MQGTYWPPT(SEQ ID NO: 202); ID NO: 203) (SEQ ID NO: 204) 45F11 RASQSVSSSLA (SEQGASTRAT (SEQ QQYINWPH ID NO: 205); ID NO: 206) (SEQ ID NO: 207) 64F9QASQDISNYLN (SEQ ID GASNLET (SEQ QQYDNFPIT NO: 208); ID NO: 209)(SEQ ID NO: 210) 72C2 RASQSVSSNLA (SEQ SASTRAS (SEQ QQYDNWPPLTID NO: 211); ID NO: 212) (SEQ ID NO: 213) 2F10 RASQSVSSSYLA (SEQGASSRAT (SEQ QQYGSSPLT ID NO: 214); ID NO: 215) (SEQ ID NO: 216) 4F11RASQDISNYLA (SEQ ID AASSLQS (SEQ LQLNSFPFT NO: 217); ID NO: 218)(SEQ ID NO: 219) 10H10 RASQGISSWLA (SEQ ID AASSLQS (SEQ QQAFSFPFTNO: 220); ID NO: 221) (SEQ ID NO: 222) 17G6 KSSQSVLYSYNNKNYVWASTRES (SEQ QQYYSTLT (SEQ A (SEQ ID NO: 223); ID NO: 224) ID NO: 225)65E11 RASQSVSSSYLA (SEQ DASSRAT (SEQ QQYGSSPLT ID NO: 226); ID NO: 227)(SEQ ID NO: 228) P02B10 SGSSSNIGSNYVY (SEQ RNNQRPS (SEQ AAWDDSLSGVVID NO: 229); ID NO: 230) (SEQ ID NO: 231) P07D03 SGSRSNIGSNYVY (SEQRNNQRPS (SEQ ASWDGSLSAVV ID NO: 232); ID NO: 233) (SEQ ID NO: 234)P08A02 SGSSSNIGSNYVY (SEQ RNNQRPS (SEQ ATWDDSLGSPV ID NO: 235);ID NO: 236) (SEQ ID NO: 237) P08E02 RASQSISRYLN (SEQ ID AASILQT (SEQ IDQQSYSTTMWT NO: 238); NO: 239) (SEQ ID NO: 240) P08F08 SGSSSNIGSNYVN (SEQGDYQRPS (SEQ ATRDDSLSGSVV ID NO: 241); ID NO: 242) (SEQ ID NO: 243)P08G02 RASQSIYDYLH (SEQ ID DASNLQS (SEQ QQSYTTPLFT NO: 244); ID NO: 245)(SEQ ID NO: 246) P12809 RASQYIGRYLN(SEQ ID  GATSLAS (SEQ QQSYSTTSPTNO: 247); ID NO: 248) (SEQ ID NO: 249) P12F02 SGSTSNIGRNYVY (SEQRTNQRPS (SEQ  AAWDDSLSGRV ID NO: 250); ID NO: 251) (SEQ ID NO: 252)P12G07 SGSSSNIGSNYVY (SEQ MNNQRPS (SEQ AAWDDSLSAVV ID NO: 253);ID NO: 254) (SEQ ID NO: 255) P13F04 SGSNSNIGTNYVS (SEQ RSSRRPS (SEQAAWDGSLSGHW ID NO: 256); ID NO: 257) V (SEQ ID NO: 258) P15D02RASQSIDTYLN (SEQ ID SASSLHS (SEQ QQSYSTTAWT NO: 259); ID NO: 260)(SEQ ID NO: 261) P16C05 RASQSIGQSLN (SEQ ID GASSLQS (SEQ QQSYSTPITNO: 262); ID NO: 263) (SEQ ID NO: 264) 10A1 RASQSISTWLA (SEQ IDKASSLES (SEQ QQYKSYSHT NO: 464); ID NO: 465) (SEQ ID NO: 466) 10E2RASQSISSWLA (SEQ ID KASSLES (SEQ QQYKSFSLT NO: 467); ID NO: 468)(SEQ ID NO: 469) 11A1 RASQSISSWLA (SEQ ID KASTLES (SEQ ID QQYNSYSYTNO: 470); NO: 471) (SEQ ID NO: 472) 11C1 RASQSVSSWLA (SEQ KASSLES (SEQQQYNTYSHT ID NO: 473); ID NO: 474) (SEQ ID NO: 475) 11D1RASQGIRNDLG (SEQ ID AASSLQS (SEQ LQDYNYPFT NO: 476); ID NO: 477)(SEQ ID NO: 478) 11E1 RASQDIDNYLA (SEQ ID AASALQS (SEQ QNYNSGPRTNO: 479); ID NO: 480) (SEQ ID NO: 481) 12A2 RASQDISNYLT (SEQ IDAASALQS (SEQ QNYNSAPRT NO: 482); ID NO: 483) (SEQ ID NO: 484) 12C4RSSQSLLHSNGYNYLD LGSNRAS (SEQ MQTLQTPFT (SEQ ID NO: 485); ID NO: 486)(SEQ ID NO: 487) 12C5 RASQGINSHLA (SEQ ID YASTLPS (SEQ ID QQLNHYPITNO: 488); NO: 489) (SEQ ID NO: 490) 12D3 RASQGISNYLA (SEQ IDAASTLHS (SEQ QKYNSAPRT NO: 491); ID NO: 492) (SEQ ID NO: 493) 12D6RASQDISNYLA (SEQ ID AASTLHS (SEQ QKYNSAPRT NO: 494); ID NO: 495)(SEQ ID NO: 496) 12D7 RASQDISSFLA (SEQ ID VASTLQS (SEQ QQLHVYPITNO: 497); ID NO: 498) (SEQ ID NO: 499) 12F5 RSSQSLLDSDDGNtYLDTLSYRAS (SEQ MQRIEFPFT (SEQ ID NO: 500); ID NO: 501) (SEQ ID NO: 502)12H4 RASQTISTWLA (SEQ ID KASNLES (SEQ QQYQTFSHT NO: 503); ID NO: 504)(SEQ ID NO: 505) 8C8 RASQGISNYLA (SEQ ID AASTLQS (SEQ QKYNSAPLTNO: 506); ID NO: 507) (SEQ ID NO: 508) 8F7 RSSQTLVHSNGYNYLN LGSNRAS (SEQMQAIQTPYT (SEQ ID NO: 509); ID NO: 510) (SEQ ID NO: 511) 8F8RASQSISSWLA (SEQ ID KASNLES (SEQ QQYNSYSCT NO: 512); ID NO: 513)(SEQ ID NO: 514) 9D8 QASQDINNYLH (SEQ ID DASDWET (SEQ QQYDHLPITNO: 515); ID NO: 516) (SEQ ID NO: 517) 9E10 QASQDISNYLH (SEQ IDDASDLET (SEQ QQYDHLPIT NO: 518); ID NO: 519) (SEQ ID NO: 520) 9E5QASQDISNYLH (SEQ ID DASDLET (SEQ QQYDHLPIT NO: 521); ID NO: 522)(SEQ ID NO: 523) 9F4 QASQDISNYLN (SEQ ID DASNLET (SEQ QQYDNLPYTNO: 524); ID NO: 525) (SEQ ID NO: 526) 9F8 RSSQSLLYSNGYNYLD LNSNRAS (SEQMQALQTPLT (SEQ ID NO: 527); ID NO: 528) (SEQ ID NO: 529)

In some embodiments, the present invention provides an antibody thatbinds to CD70 and competes with the antibody as described herein,including 31H1, 6362, 40E3, 42C3, 45F11, 64F9, 72C2, 2F10, 4F11, 10H10,17G6, 65E11, P02B10, P07D03, P08A02, P08E02, P08F08, P08G02, P12B09,P12F02, P12G07, P13F04, P15D02, P16C05, 10A1, 10E2, 11A1, 11C1, 11D1,11E1, 12A2, 12C4, 12C5, 12D3, 12D6, 12D7, 12F5, 12H4, 8C8, 8F7, 8F8,9D8, 9E10, 9E5, 9F4 or 9F8.

In some embodiments, the invention also provides CDR portions ofantibodies to CD70 antibodies based on CDR contact regions. CDR contactregions are regions of an antibody that imbue specificity to theantibody for an antigen. In general, CDR contact regions include theresidue positions in the CDRs and Vernier zones which are constrained inorder to maintain proper loop structure for the antibody to bind aspecific antigen. See, e.g., Makabe et al., J. Biol. Chem.,283:1156-1166, 2007. Determination of CDR contact regions is well withinthe skill of the art.

The binding affinity (Ko) of the CD70 antibody as described herein toCD70 (such as human CD70 (e.g., (SEQ ID NO: 278)) can be about 0.001 toabout 5000 nM. In some embodiments, the binding affinity is about any of5000 nM, 4500 nM, 4000 nM, 3500 nM, 3000 nM, 2500 nM, 2000 nM, 1789 nM,1583 nM, 1540 nM, 1500 nM, 1490 nM, 1064 nM, 1000 nM, 933 nM, 894 nM,750 nM, 705 nM, 678 nM, 532 nM, 500 nM, 494 nM, 400 nM, 349 nM, 340 nM,353 nM, 300 nM, 250 nM, 244 nM, 231 nM, 225 nM, 207 nM, 200 nM, 186 nM,172 nM, 136 nM, 113 nM, 104 nM, 101 nM, 100 nM, 90 nM, 83 nM, 79 nM, 74nM, 54 nM, 50 nM, 45 nM, 42 nM, 40 nM, 35 nM, 32 nM, 30 nM, 25 nM, 24nM, 22 nM, 20 nM, 19 nM, 18 nM, 17 nM, 16 nM, 15 nM, 12 nM, 10 nM, 9 nM,8 nM, 7.5 nM, 7 nM, 6.5 nM, 6 nM, 5.5 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM,0.5 nM, 0.3 nM, 0.1 nM, 0.01 nM, or 0.001 nM. In some embodiments, thebinding affinity is less than about any of 5000 nM, 4000 nM, 3000 nM,2000 nM, 1000 nM, 900 nM, 800 nM, 250 nM, 200 nM, 100 nM, 50 nM, 30 nM,20 nM, 10 nM, 7.5 nM, 7 nM, 6.5 nM, 6 nM, 5 nM, 4.5 nM, 4 nM, 3.5 nM, 3nM, 2.5 nM, 2 nM, 1.5 nM, 1 nM, or 0.5 nM.

Bispecific antibodies, monoclonal antibodies that have bindingspecificities for at least two different antigens, can be prepared usingthe antibodies disclosed herein. Methods for making bispecificantibodies are known in the art (see, e.g., Suresh et al., Methods inEnzymology 121:210, 1986). Traditionally, the recombinant production ofbispecific antibodies was based on the coexpression of twoimmunoglobulin heavy chain-light chain pairs, with the two heavy chainshaving different specificities (Millstein and Cuello, Nature 305,537-539, 1983). Accordingly, in one aspect, provided is a bispecificantibody wherein the bispecific antibody is a full-length humanantibody, comprising a first antibody variable domain of the bispecificantibody specifically binding to a target antigen (e.g., CD70), andcomprising a second antibody variable domain of the bispecific antibodycapable of recruiting the activity of a human immune effector cell byspecifically binding to an effector antigen located on the human immuneeffector cell.

The human immune effector cell can be any of a variety of immuneeffector cells known in the art. For example, the immune effector cellcan be a member of the human lymphoid cell lineage, including, but notlimited to, a T cell (e.g., a cytotoxic T cell), a B cell, and a naturalkiller (NK) cell. The immune effector cell can also be, for examplewithout limitation, a member of the human myeloid lineage, including,but not limited to, a monocyte, a neutrophilic granulocyte, and adendritic cell. Such immune effector cells may have either a cytotoxicor an apoptotic effect on a target cell or other desired effect uponactivation by binding of an effector antigen.

The effector antigen is an antigen (e.g., a protein or a polypeptide)that is expressed on the human immune effector cell. Examples ofeffector antigens that can be bound by the heterodimeric protein (e.g.,a heterodimeric antibody or a bispecific antibody) include, but are notlimited to, human CD3 (or CD3 (Cluster of Differentiation) complex),CD16, NKG2D, NKp46, CD2, CD28, CD25, CD64, and CD89.

The target cell can be a cell that is native or foreign to humans. In anative target cell, the cell may have been transformed to be a malignantcell or pathologically modified (e.g., a native target cell infectedwith a virus, a plasmodium, or a bacterium). In a foreign target cell,the cell is an invading pathogen, such as a bacterium, a plasmodium, ora virus.

The target antigen is expressed on a target cell in a diseased condition(e.g., an inflammatory disease, a proliferative disease (e.g., cancer),an immunological disorder, a neurological disease, a neurodegenerativedisease, an autoimmune disease, an infectious disease (e.g., a viralinfection or a parasitic infection), an allergic reaction, agraft-versus-host disease or a host-versus-graft disease). A targetantigen is not effector antigen. In some embodiments, the target antigenis CD70.

In some embodiments, provided is a bispecific antibody, wherein thebispecific antibody is a full-length antibody, comprising a firstantibody variable domain of the bispecific antibody specifically bindingto a target antigen, and comprising a second antibody variable domain ofthe bispecific antibody capable of recruiting the activity of a humanimmune effector cell by specifically binding to an effector antigenlocated on the human immune effector cell, wherein the first antibodyvariable domain comprises a heavy chain variable (VH) region comprisinga VH CDR1, VH CDR2, and VH CDR3 of the VH sequence shown in SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38,40, 42, 44, 46, 48, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307,309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329 or 331; or a lightchain variable (VL) region comprising VL CDR1, VL CDR2, and VL CDR3 ofthe VL sequence shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19,21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 288, 290, 292,294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320,322, 324, 326, 328 or 330.

In some embodiments, provided is a bispecific antibody, wherein thebispecific antibody is a full-length antibody, comprising a firstantibody variable domain of the bispecific antibody specifically bindingto a target antigen, and comprising a second antibody variable domain ofthe bispecific antibody capable of recruiting the activity of a humanimmune effector cell by specifically binding to an effector antigenlocated on the human immune effector cell, wherein the first antibodyvariable domain comprises (a) a heavy chain variable (VH) regioncomprising (i) a VH complementarity determining region one (CDR1)comprising the sequence shown in SEQ ID NO: 49, 50, 51, 55, 56, 57, 61,62, 63, 67, 68, 69, 73, 74, 75, 79, 80, 81, 85, 86, 87, 91, 92, 93, 97,98, 99, 103, 104, 105, 109, 110, 111, 115, 116, 117, 121, 122, 123, 127,128, 129, 133, 134, 135, 139, 140, 141, 145, 146, 147, 151, 152, 153,157, 158, 159, 163, 164, 165, 169, 170, 171, 175, 176, 177, 181, 182,183, 187, 188, 189, 332, 333, 334, 338, 339, 340, 344, 345, 346, 350,351, 352, 356, 357, 358, 362, 363, 364, 368, 369, 370, 374, 375, 376,380, 381, 382, 386, 387, 388, 392, 393, 394, 398, 399, 400, 404, 405,406, 410, 411, 412, 416, 437, 418, 422, 423, 424, 428, 429, 430, 434,435, 436, 440, 441, 442, 446, 447, 448, 452, 453, 454, 458, 459 or 460;(ii) a VH CDR2 comprising the sequence shown in SEQ ID NO: 52, 53, 58,59, 64, 65, 70, 71, 76, 77, 82, 83, 88, 89, 94, 95, 100, 101, 106, 107,112, 113, 118, 119, 124, 125, 130, 131, 136, 137, 142, 143, 148, 149,154, 155, 160, 161, 166, 167, 172, 173, 178, 179, 184, 185, 190, 191,335, 336, 341, 342, 347, 348, 353, 354, 359, 360, 365, 366, 371, 372,377, 378, 383, 384, 389, 390, 395, 396, 401, 402, 407, 408, 413, 414,419, 420, 425, 426, 431, 432, 437, 438, 443, 444, 449, 450, 455, 456,461 or 462; and iii) a VH CDR3 comprising the sequence shown in SEQ IDNO: 54, 60, 66, 72, 78, 84, 90, 96, 102, 108, 114, 120, 126, 132, 138,144, 150, 156, 162, 168, 174, 180, 186, 192, 337, 343, 349, 355, 361,367, 373, 379, 385, 391, 397, 403, 409, 415, 421, 427, 433, 439, 445,451, 457 or 463; or a light chain variable (VL) region comprising (i) aVL CDR1 comprising the sequence shown in SEQ ID NO: 193, 196, 199, 202,205, 208, 211, 214, 217, 220, 223, 226, 229, 232, 235, 238, 241, 244,247, 250, 253, 256, 259, 262, 464, 467, 470, 473, 476, 479, 482, 485,488, 491, 494, 497, 500, 503, 506, 509, 512, 515, 518, 521, 524 or 527;(ii) a VL CDR2 comprising the sequence shown in SEQ ID NO: 194, 197,200, 203, 206, 209, 212, 215, 218, 221, 224, 227, 230, 233, 236, 239,242, 245, 248, 251, 254, 257, 260, 263, 465, 468, 471, 474, 477, 480,483, 486, 489, 492, 495, 498, 501, 504, 507, 510, 513, 516, 519, 522,525 or 528; and (iii) a VL CDR3 comprising the sequence shown in SEQ IDNO: 195, 198, 201, 204, 207, 210, 213, 216, 219, 222, 225, 228, 231,234, 237, 240, 243, 246, 249, 252, 255, 258, 261, 264, 466, 469, 472,475, 478, 481, 484, 487, 490, 493, 496, 499, 502, 505, 508, 511, 514,517, 520, 523, 526 or 529.

In some embodiments, the second antibody variable domain comprises aheavy chain variable (VH) region comprising a VH CDR1, VH CDR2, and VHCDR3 of the VH sequence shown in SEQ ID NO: 266; or a light chainvariable (VL) region comprising VL CDR1, VL CDR2, and VL CDR3 of the VLsequence shown in SEQ ID NO: 265.

In some embodiments, the second antibody variable domain comprises (a) aheavy chain variable (VH) region comprising (i) a VH complementarydetermining region one (CDR1) comprising the sequence shown in SEQ IDNO: 267, 268, or 269; (ii) a VH CDR2 comprising the sequence shown inSEQ ID NO: 270 or 271; and iii) a VH CDR3 comprising the sequence shownin SEQ ID NO: 272; or (b) a light chain variable (VL) region comprising(i) a VL CDR1 comprising the sequence shown in SEQ ID NO: 273; (ii) a VLCDR2 comprising the sequence shown in SEQ ID NO: 274; and (iii) a VLCDR3 comprising the sequence shown in SEQ ID NO: 275.

Table 3 shows the specific amino acid and nucleic acid sequences of thesecond antibody variable domain, which is specific to CD3. In Table 3,the underlined sequences are CDR sequences according to Kabat and inbold according to Chothia.

TABLE 3 mAb Light Chain Heavy Chain h2B4_ DIVMTQSPDSLAVSLGERATINCEVQLVESGGGLVQPGGSLRLSCA HNPS_ KSSQSLFNVRSRKNYLA WYQQK ASGFTFSDYYMTWVRQAPGKGLE VL_TK PGQPPKLLIS WASTRES GVPDRF WVAFIRNRARGYTSDHNPSVKGRSGSGSGTDFTLTISSLQAEDVAV FTISRDNAKNSLYLQMNSLRAEDT YYC KQSYDLFT FGSGTKLEIKAVYYCAR DRPSYYVLDY WGQGTT (SEQ ID NO: 265) VTVSS (SEQ ID NO: 266) h2B4_GACATTGTGATGACTCAATCCC GAAGTCCAACTTGTCGAATCGGG HNPS_CCGACTCCCTGGCTGTGTCCCT AGGAGGCCTTGTGCAACCCGGT VL_TKCGGCGAACGCGCAACTATCAAC GGATCCCTGAGGCTGTCATGCG TGTAAAAGCAGCCAGTCCCTGTCGGCCTCGGGCTTCACCTTTTCC TCAACGTCCGGTCGAGGAAGAA GATTACTACATGACCTGGGTCAGCTACCTGGCCTGGTATCAGCAG ACAGGCCCCTGGAAAGGGGTTG AAACCTGGGCAGCCGCCGAAGGAATGGGTGGCATTCATCCGGA CTTCTGATCTCATGGGCCTCAA ATAGAGCCCGCGGATACACTTCCCTCGGGAAAGCGGAGTGCCAG GACCACAACCCCAGCGTGAAGG ATAGATTCTCCGGATCTGGCTCGGCGGTTCACCATTAGCCGCGA CGGAACCGACTTCACCCTGACG CAACGCCAAGAACTCCCTCTACCATTTCGAGCTTGCAAGCGGAGG TCCAAATGAACAGCCTGCGGGC ATGTGGCCGTGTACTACTGCAAGGAGGATACCGCTGTGTACTACT GCAGTCCTACGACCTCTTCACC GCGCCCGCGACCGGCCGTCCTATTTGGTTCGGGCACCAAGCTGG CTATGTGCTGGACTACTGGGGC AGATCAAA (SEQ ID NO: 276)CAGGGTACTACGGTCACCGTCT CCTCA (SEQ ID NO: 277)

Table 4 shows the examples of CDR sequences of the second antibodyvariable domain, which is specific to CD3.

TABLE 4 Heavy Chain mAb CDRH1 CDRH2 CDRH3 h2B4_ SDYYMT (SEQ IDRNRARGYT (SEQ ID NO: DRPSYYVLDY HNPS_VL_ NO: 267) (Kabat); 270) (Kabat)(SEQ ID NO: 272) TK GFTFSDY (SEQ ID FIRNRARGYTSDHNPSVKGNO: 268) (Chothia); (SEQ ID NO: 271) GFTFSDYYMT (SEQ (Extended)ID NO: 269) (Extended) Light Chain mAb CDRH1 CDRH2 CDRH3 h2B4_KSSQSLFNVRSRKN WASTRES KQSYDLFT HNPS_VL_ YLA (SEQ ID NO: 274)(SEQ ID NO: 275) TK (SEQ ID NO: 273)

In some embodiments, a bispecific antibody provided herein whichcontains a CD3-specific variable domain having an anti-CD3 sequence asprovided in U.S. Publication No. 20160297885, which is herebyincorporated by reference for all purposes.

According to one approach to making bispecific antibodies, antibodyvariable domains with the desired binding specificities(antibody-antigen combining sites) are fused to immunoglobulin constantregion sequences. The fusion preferably is with an immunoglobulin heavychain constant region, comprising at least part of the hinge, CH2 andCH3 regions. It is preferred to have the first heavy chain constantregion (CH1), containing the site necessary for light chain binding,present in at least one of the fusions. DNAs encoding the immunoglobulinheavy chain fusions and, if desired, the immunoglobulin light chain, areinserted into separate expression vectors, and are cotransfected into asuitable host organism. This provides for great flexibility in adjustingthe mutual proportions of the three polypeptide fragments in embodimentswhen unequal ratios of the three polypeptide chains used in theconstruction provide the optimum yields. It is, however, possible toinsert the coding sequences for two or all three polypeptide chains inone expression vector when the expression of at least two polypeptidechains in equal ratios results in high yields or when the ratios are ofno particular significance.

In another approach, the bispecific antibodies are composed of a hybridimmunoglobulin heavy chain with a first binding specificity in one arm,and a hybrid immunoglobulin heavy chain-light chain pair (providing asecond binding specificity) in the other arm. This asymmetric structure,with an immunoglobulin light chain in only one half of the bispecificmolecule, facilitates the separation of the desired bispecific compoundfrom unwanted immunoglobulin chain combinations. This approach isdescribed in PCT Publication No. WO 94/04690.

In another approach, the bispecific antibodies are composed of aminoacid modification in the first hinge region in one arm, and thesubstituted/replaced amino acid in the first hinge region has anopposite charge to the corresponding amino acid in the second hingeregion in another arm. This approach is described in InternationalPatent Application No. PCT/US2011/036419 (WO2011/143545).

In another approach, the formation of a desired heteromultimeric orheterodimeric protein (e.g., bispecific antibody) is enhanced byaltering or engineering an interface between a first and a secondimmunoglobulin-like Fc region (e.g., a hinge region or a CH3 region). Inthis approach, the bispecific antibodies may be composed of a CH3region, wherein the CH3 region comprises a first CH3 polypeptide and asecond CH3 polypeptide which interact together to form a CH3 interface,wherein one or more amino acids within the CH3 interface destabilizehomodimer formation and are not electrostatically unfavorable tohomodimer formation. This approach is described in International PatentApplication No. PCT/US2011/036419 (WO2011/143545).

In another approach, the bispecific antibodies can be generated using aglutamine-containing peptide tag engineered to the antibody directed toan epitope (e.g., CD70) in one arm and another peptide tag (e.g., aLys-containing peptide tag or a reactive endogenous Lys) engineered to asecond antibody directed to a second epitope in another arm in thepresence of transglutaminase. This approach is described inInternational Patent Application No. PCT/IB2011/054899 (WO2012/059882).

In some embodiments, the heterodimeric protein (e.g., bispecificantibody) as described herein comprises a full-length human antibody,wherein a first antibody variable domain of the bispecific antibodyspecifically binding to a target antigen (e.g., CD70), and comprising asecond antibody variable domain of the bispecific antibody capable ofrecruiting the activity of a human immune effector cell by specificallybinding to an effector antigen (e.g., CD3) located on the human immuneeffector cell, wherein the first and second antibody variable domain ofthe heterodimeric protein comprise amino acid modifications at positions223, 225, and 228 (e.g., (C223E or C223R), (E225E or E225R), and (P228Eor P228R)) in the hinge region and at position 409 or 368 (e.g., K409Ror L368E (EU numbering scheme)) in the CH3 region of human IgG2 (SEQ IDNO: 279).

In some embodiments, the first and second antibody variable domains ofthe heterodimeric protein comprise amino acid modifications at positions221 and 228 (e.g., (D221R or D221E) and (P228R or P228E)) in the hingeregion and at position 409 or 368 (e.g., K409R or L368E (EU numberingscheme)) in the CH3 region of human IgG1 (SEQ ID NO: 280).

In some embodiments, the first and second antibody variable domains ofthe heterodimeric protein comprise amino acid modifications at positions228 (e.g., (P228E or P228R)) in the hinge region and at position 409 or368 (e.g., R409 or L368E (EU numbering scheme)) in the CH3 region ofhuman IgG4 (SEQ ID NO: 281).

The antibodies useful in the present invention can encompass monoclonalantibodies, polyclonal antibodies, antibody fragments (e.g., Fab, Fab′,F(ab′)2, Fv, Fc, etc.), chimeric antibodies, bispecific antibodies,heteroconjugate antibodies, single chain (ScFv), mutants thereof, fusionproteins comprising an antibody portion (e.g., a domain antibody),humanized antibodies, and any other modified configuration of theimmunoglobulin molecule that comprises an antigen recognition site ofthe required specificity, including glycosylation variants ofantibodies, amino acid sequence variants of antibodies, and covalentlymodified antibodies. The antibodies may be murine, rat, human, or anyother origin (including chimeric or humanized antibodies).

In some embodiments, the CD70 monospecific antibody or the CD70bispecific antibody (e.g., CD70-CD3) as described herein is a monoclonalantibody. For example, the CD70 monospecific antibody is a humanmonoclonal antibody. In another example, the CD70 arm of the CD70-CD3bispecific antibody is a human monoclonal antibody, and the CD3 arm ofthe CD70-CD3 bispecific antibody is a humanized monoclonal antibody.

In some embodiments, the antibody comprises a modified constant region,such as, for example without limitation, a constant region that hasincreased potential for provoking an immune response. For example, theconstant region may be modified to have increased affinity to an Fcgamma receptor such as, e.g., FcγRI, FcγRIIA, or FcγIII.

In some embodiments, the antibody comprises a modified constant region,such as a constant region that is immunologically inert, that is, havinga reduced potential for provoking an immune response. In someembodiments, the constant region is modified as described in Eur. J.Immunol., 29:2613-2624, 1999; PCT Application No. PCT/GB99/01441; or UKPatent Application No. 98099518. The Fc can be human IgG1, human IgG2,human IgG3, or human IgG4. The Fc can be human IgG2 containing themutation A330P331 to S330S331 (IgG2Δa), in which the amino acid residuesare numbered with reference to the wild type IgG2 sequence. Eur. J.Immunol., 29:2613-2624, 1999. In some embodiments, the antibodycomprises a constant region of IgG4 comprising the following mutations(Armour et al., Molecular Immunology 40 585-593, 2003): E233F234L235 toP233V234A235 (IgG4Δc), in which the numbering is with reference to wildtype IgG4. In yet another embodiment, the Fc is human IgG4 E233F234L235to P233V234A235 with deletion G236 (IgG4Δb). In another embodiment, theFc is any human IgG4 Fc (IgG4, IgG4Δb or IgG4Δc) containing hingestabilizing mutation S228 to P228 (Aalberse et al., Immunology 105,9-19, 2002). In another embodiment, the Fc can be aglycosylated Fc.

In some embodiments, the constant region is aglycosylated by mutatingthe oligosaccharide attachment residue (such as Asn297) or flankingresidues that are part of the glycosylation recognition sequence in theconstant region. In some embodiments, the constant region isaglycosylated for N-linked glycosylation enzymatically. The constantregion may be aglycosylated for N-linked glycosylation enzymatically orby expression in a glycosylation deficient host cell.

In some embodiments, the constant region has a modified constant regionthat removes or reduces Fc gamma receptor binding. For example, the Fccan be human IgG2 containing the mutation D265, in which the amino acidresidues are numbered with reference to the wild type IgG2 sequence (SEQID NO: 279). Accordingly, in some embodiments, the constant region has amodified constant region having the sequence shown in SEQ ID NO: 282:ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCRVRCPRCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVQFNVVYVDGVEVHNAKTKPREEQFNSTFRWSVLTWHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSRLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK. And the nucleic acid encoding the sequenceshown in SEQ ID NO: 282 is shown in SEQ ID NO: 283.

In some embodiments, the constant region has a modified constant regionhaving the sequence shown in SEQ ID NO: 284:ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCEVECPECPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVQFNVVYVDGVEVHNAKTKPREEQFNSTFRWSVLTWHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCEVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK. And the nucleic acid encoding the sequenceshown in SEQ ID NO: 284 is shown in SEQ ID NO: 285.

The amino acid of the human Kappa constant region is shown in SEQ ID NO:286: GTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC. And the nucleic acidencoding the sequence of SEQ ID NO: 286 is shown in SEQ ID NO: 287.

One way of determining binding affinity of antibodies to CD70 is bymeasuring binding affinity of the bivalent antibody to monomeric CD70protein. The affinity of an CD70 antibody can be determined by surfaceplasmon resonance (Biacore™3000™ surface plasmon resonance (SPR) system,Biacore™, INC, Piscataway N.J.) equipped with pre-immobilized anti-mouseFc or anti-human Fc using HBS-EP running buffer (0.01M HEPES, pH 7.4,0.15 NaCl, 3 mM EDTA, 0.005% v/v Surfactant P20). Monomeric 8-histidinetagged human CD70 extracellular domain (SEQ ID NO: 530) can be dilutedinto HBS-EP buffer to a concentration of less than 0.5 μg/mL andinjected across the individual chip channels using variable contacttimes, to achieve two ranges of antigen density, either 50-200 responseunits (RU) for detailed kinetic studies or 800-1,000 RU for screeningassays. Regeneration studies have shown that 25 mM NaOH in 25% v/vethanol effectively removes the bound CD70 protein while keeping theactivity of CD70 antibodies on the chip for over 200 injections.Typically, serial dilutions (spanning concentrations of 0.1-10×estimated Ko) of purified 8-histidine tagged CD70 samples (SEQ ID NO:530) are injected for 1 min at 100 μL/minute and dissociation times ofup to 2 hours are allowed. The concentrations of the CD70 proteins aredetermined by absorbance at 280 nm based on sequence specific extinctioncoefficient of the 8-histidine tagged CD70 protein (SEQ ID NO: 530).Kinetic association rates (k_(on) or k_(a)) and dissociation rates(k_(off) or k_(d)) are obtained simultaneously by fitting the dataglobally to a 1:1 Langmuir binding model (Karlsson, R. Roos, H.Fagerstam, L. Petersson, B. (1994). Methods Enzymology 6. 99-110) usingthe BIAevaluation program. Equilibrium dissociation constant (Ko) valuesare calculated as k_(off)/k_(on). This protocol is suitable for use indetermining binding affinity of an antibody to any monomeric CD70,including human CD70, CD70 of another mammal (such as mouse CD70, ratCD70, or primate CD70), as well as different forms of CD70 (e.g.,glycosylated CD70). Binding affinity of an antibody is generallymeasured at 25° C., but can also be measured at 37° C.

The antibodies as described herein may be made by any method known inthe art. For the production of hybridoma cell lines, the route andschedule of immunization of the host animal are generally in keepingwith established and conventional techniques for antibody stimulationand production, as further described herein. General techniques forproduction of human and mouse antibodies are known in the art or aredescribed herein.

It is contemplated that any mammalian subject including humans orantibody producing cells therefrom can be manipulated to serve as thebasis for production of mammalian, including human and hybridoma celllines. Typically, the host animal is inoculated intraperitoneally,intramuscularly, orally, subcutaneously, intraplantar, or intradermallywith an amount of immunogen, including as described herein.

Hybridomas can be prepared from the lymphocytes and immortalized myelomacells using the general somatic cell hybridization technique of Kohler,B. and Milstein, C., Nature 256:495-497, 1975 or as modified by Buck, D.W., et al., In Vitro, 18:377-381, 1982. Available myeloma lines,including but not limited to X63-Ag8.653 and those from the SalkInstitute, Cell Distribution Center, San Diego, Calif., USA, may be usedin the hybridization. Generally, the technique involves fusing myelomacells and lymphoid cells using a fusogen such as polyethylene glycol, orby electrical means well known to those skilled in the art. After thefusion, the cells are separated from the fusion medium and grown in aselective growth medium, such as hypoxanthine-aminopterin-thymidine(HAT) medium, to eliminate unhybridized parent cells. Any of the mediadescribed herein, supplemented with or without serum, can be used forculturing hybridomas that secrete monoclonal antibodies. As anotheralternative to the cell fusion technique, EBV immortalized B cells maybe used to produce the monoclonal antibodies of the subject invention.The hybridomas are expanded and subcloned, if desired, and supernatantsare assayed for anti-immunogen activity by conventional immunoassayprocedures (e.g., radioimmunoassay, enzyme immunoassay, or fluorescenceimmunoassay).

Hybridomas that may be used as source of antibodies encompass allderivatives, progeny cells of the parent hybridomas that producemonoclonal antibodies specific for CD70, or portions thereof.

Hybridomas that produce such antibodies may be grown in vitro or in vivousing known procedures. The monoclonal antibodies may be isolated fromthe culture media or body fluids, by conventional immunoglobulinpurification procedures such as ammonium sulfate precipitation, gelelectrophoresis, dialysis, chromatography, and ultrafiltration, ifdesired. Undesired activity, if present, can be removed, for example, byrunning the preparation over adsorbents made of the immunogen attachedto a solid phase and eluting or releasing the desired antibodies off theimmunogen. Immunization of a host animal with cells expressing humanCD70, a human CD70 protein, or a fragment containing the target aminoacid sequence conjugated to a protein that is immunogenic in the speciesto be immunized, e.g., keyhole limpet hemocyanin, serum albumin, bovinethyroglobulin, or soybean trypsin inhibitor using a bifunctional orderivatizing agent, for example, maleimidobenzoyl sulfosuccinimide ester(conjugation through cysteine residues), N-hydroxysuccinimide (throughlysine residues), glutaraldehyde, succinic anhydride, SOCl₂, orR¹N═C═NR, where R and R¹ are different alkyl groups, can yield apopulation of antibodies (e.g., monoclonal antibodies).

If desired, the antibody (monoclonal or polyclonal) of interest may besequenced and the polynucleotide sequence may then be cloned into avector for expression or propagation. The sequence encoding the antibodyof interest may be maintained in vector in a host cell and the host cellcan then be expanded and frozen for future use. Production ofrecombinant monoclonal antibodies in cell culture can be carried outthrough cloning of antibody genes from B cells by means known in theart. See, e.g. Tiller et al., J. Immunol. Methods 329, 112, 2008; U.S.Pat. No. 7,314,622.

In an alternative, the polynucleotide sequence may be used for geneticmanipulation to “humanize” the antibody or to improve the affinity, orother characteristics of the antibody. For example, the constant regionmay be engineered to more nearly resemble human constant regions toavoid immune response if the antibody is used in clinical trials andtreatments in humans. It may be desirable to genetically manipulate theantibody sequence to obtain greater affinity to CD70 and greaterefficacy in inhibiting CD70.

There are four general steps to humanize a monoclonal antibody. Theseare: (1) determining the nucleotide and predicted amino acid sequence ofthe starting antibody light and heavy variable domains (2) designing thehumanized antibody, i.e., deciding which antibody framework region touse during the humanizing process (3) the actual humanizingmethodologies/techniques and (4) the transfection and expression of thehumanized antibody. See, for example, U.S. Pat. Nos. 4,816,567;5,807,715; 5,866,692; 6,331,415; 5,530,101; 5,693,761; 5,693,762;5,585,089; and 6,180,370.

A number of “humanized” antibody molecules comprising an antigen bindingsite derived from a non-human immunoglobulin have been described,including chimeric antibodies having rodent or modified rodent V regionsand their associated CDRs fused to human constant regions. See, forexample, Winter et al. Nature 349:293-299, 1991, Lobuglio et al. Proc.Nat. Acad. Sci. USA 86:4220-4224, 1989, Shaw et al. J Immunol.138:4534-4538, 1987, and Brown et al. Cancer Res. 47:3577-3583, 1987.Other references describe rodent CDRs grafted into a human supportingframework region (FR) prior to fusion with an appropriate human antibodyconstant region. See, for example, Riechmann et al. Nature 332:323-327,1988, Verhoeyen et al. Science 239:1534-1536, 1988, and Jones et al.Nature 321:522-525, 1986. Another reference describes rodent CDRssupported by recombinantly engineered rodent framework regions. See, forexample, European Patent Publication No. 0519596. These “humanized”molecules are designed to minimize unwanted immunological responsetoward rodent anti-human antibody molecules which limits the durationand effectiveness of therapeutic applications of those moieties in humanrecipients. For example, the antibody constant region can be engineeredsuch that it is immunologically inert (e.g., does not trigger complementlysis). See, e.g. PCT Publication No. PCT/GB99/01441; UK PatentApplication No. 9809951.8. Other methods of humanizing antibodies thatmay also be utilized are disclosed by Daugherty et al., Nucl. Acids Res.19:2471-2476, 1991, and in U.S. Pat. Nos. 6,180,377; 6,054,297;5,997,867; 5,866,692; 6,210,671; and 6,350,861; and in PCT PublicationNo. WO 01/27160.

The general principles related to humanized antibodies discussed aboveare also applicable to customizing antibodies for use, for example, indogs, cats, primate, equines and bovines. Further, one or more aspectsof humanizing an antibody described herein may be combined, e.g., CDRgrafting, framework mutation and CDR mutation.

In one variation, fully human antibodies may be obtained by usingcommercially available mice that have been engineered to expressspecific human immunoglobulin proteins. Transgenic animals that aredesigned to produce a more desirable (e.g., fully human antibodies) ormore robust immune response may also be used for generation of humanizedor human antibodies. Examples of such technology are Xenomouse™ fromAbgenix, Inc. (Fremont, Calif.) and HuMAb-Mouse® and TC Mouse™ fromMedarex, Inc. (Princeton, N.J.).

In an alternative, antibodies may be made recombinantly and expressedusing any method known in the art. In another alternative, antibodiesmay be made recombinantly by phage display technology. See, for example,U.S. Pat. Nos. 5,565,332; 5,580,717; 5,733,743; and 6,265,150; andWinter et al., Annu. Rev. Immunol. 12:433-455, 1994. Alternatively, thephage display technology (McCafferty et al., Nature 348:552-553, 1990)can be used to produce human antibodies and antibody fragments in vitro,from immunoglobulin variable (V) domain gene repertoires fromunimmunized donors. According to this technique, antibody V domain genesare cloned in-frame into either a major or minor coat protein gene of afilamentous bacteriophage, such as M13 or fd, and displayed asfunctional antibody fragments on the surface of the phage particle.Because the filamentous particle contains a single-stranded DNA copy ofthe phage genome, selections based on the functional properties of theantibody also result in selection of the gene encoding the antibodyexhibiting those properties. Thus, the phage mimics some of theproperties of the B cell. Phage display can be performed in a variety offormats; for review see, e.g., Johnson, Kevin S. and Chiswell, David J.,Current Opinion in Structural Biology 3:564-571, 1993. Several sourcesof V-gene segments can be used for phage display. Clackson et al.,Nature 352:624-628, 1991, isolated a diverse array of anti-oxazoloneantibodies from a small random combinatorial library of V genes derivedfrom the spleens of immunized mice. A repertoire of V genes fromunimmunized human donors can be constructed and antibodies to a diversearray of antigens (including self-antigens) can be isolated essentiallyfollowing the techniques described by Mark et al., J. Mol. Biol.222:581-597, 1991, or Griffith et al., EMBO J. 12:725-734, 1993. In anatural immune response, antibody genes accumulate mutations at a highrate (somatic hypermutation). Some of the changes introduced will conferhigher affinity, and B cells displaying high-affinity surfaceimmunoglobulin are preferentially replicated and differentiated duringsubsequent antigen challenge. This natural process can be mimicked byemploying the technique known as “chain shuffling.” (Marks et al.,Bio/Technol. 10:779-783, 1992). In this method, the affinity of“primary” human antibodies obtained by phage display can be improved bysequentially replacing the heavy and light chain V region genes withrepertoires of naturally occurring variants (repertoires) of V domaingenes obtained from unimmunized donors. This technique allows theproduction of antibodies and antibody fragments with affinities in thepM-nM range. A strategy for making very large phage antibody repertoires(also known as “the mother-of-all libraries”) has been described byWaterhouse et al., Nucl. Acids Res. 21:2265-2266, 1993. Gene shufflingcan also be used to derive human antibodies from rodent antibodies,where the human antibody has similar affinities and specificities to thestarting rodent antibody. According to this method, which is alsoreferred to as “epitope imprinting”, the heavy or light chain V domaingene of rodent antibodies obtained by phage display technique isreplaced with a repertoire of human V domain genes, creatingrodent-human chimeras. Selection on antigen results in isolation ofhuman variable regions capable of restoring a functional antigen bindingsite, i.e., the epitope governs (imprints) the choice of partner. Whenthe process is repeated in order to replace the remaining rodent Vdomain, a human antibody is obtained (see PCT Publication No. WO93/06213). Unlike traditional humanization of rodent antibodies by CDRgrafting, this technique provides completely human antibodies, whichhave no framework or CDR residues of rodent origin.

Antibodies may be made recombinantly by first isolating the antibodiesand antibody producing cells from host animals, obtaining the genesequence, and using the gene sequence to express the antibodyrecombinantly in host cells (e.g., CHO cells). Another method which maybe employed is to express the antibody sequence in plants (e.g.,tobacco) or transgenic milk. Methods for expressing antibodiesrecombinantly in plants or milk have been disclosed. See, for example,Peeters, et al. Vaccine 19:2756, 2001; Lonberg, N. and D. Huszar Int.Rev. Immunol 13:65, 1995; and Pollock, et al., J Immunol Methods231:147, 1999. Methods for making derivatives of antibodies, e.g.,humanized, single chain, etc. are known in the art.

Immunoassays and flow cytometry sorting techniques such as fluorescenceactivated cell sorting (FACS) can also be employed to isolate antibodiesthat are specific for CD70, or tumor antigens of interest.

The antibodies as described herein can be bound to many differentcarriers. Carriers can be active or inert. Examples of well-knowncarriers include polypropylene, polystyrene, polyethylene, dextran,nylon, amylases, glass, natural and modified celluloses,polyacrylamides, agaroses, and magnetite. The nature of the carrier canbe either soluble or insoluble for purposes of the invention. Thoseskilled in the art will know of other suitable carriers for bindingantibodies, or will be able to ascertain such, using routineexperimentation. In some embodiments, the carrier comprises a moietythat targets the myocardium.

DNA encoding the monoclonal antibodies is readily isolated and sequencedusing conventional procedures (e.g., by using oligonucleotide probesthat are capable of binding specifically to genes encoding the heavy andlight chains of the monoclonal antibodies). The hybridoma cells serve asa preferred source of such DNA. Once isolated, the DNA may be placedinto expression vectors (such as expression vectors disclosed in PCTPublication No. WO 87/04462), which are then transfected into host cellssuch as E. coli cells, simian COS cells, Chinese hamster ovary (CHO)cells, or myeloma cells that do not otherwise produce immunoglobulinprotein, to obtain the synthesis of monoclonal antibodies in therecombinant host cells. See, e.g., PCT Publication No. WO87/04462. TheDNA also may be modified, for example, by substituting the codingsequence for human heavy and light chain constant regions in place ofthe homologous murine sequences, Morrison et al., Proc. Nat. Acad. Sci.81:6851, 1984, or by covalently joining to the immunoglobulin codingsequence all or part of the coding sequence for a non-immunoglobulinpolypeptide. In that manner, “chimeric” or “hybrid” antibodies areprepared that have the binding specificity of a monoclonal antibodyherein.

The CD70 antibodies as described herein can be identified orcharacterized using methods known in the art, whereby reduction of CD70expression levels are detected or measured. In some embodiments, an CD70antibody is identified by incubating a candidate agent with CD70 andmonitoring binding or attendant reduction of CD70 expression levels. Thebinding assay may be performed with purified CD70 polypeptide(s), orwith cells naturally expressing, or transfected to express, CD70polypeptide(s). In one embodiment, the binding assay is a competitivebinding assay, where the ability of a candidate antibody to compete witha known CD70 antibody for CD70 binding is evaluated. The assay may beperformed in various formats, including the ELISA format.

Following initial identification, the activity of a candidate CD70antibody can be further confirmed and refined by bioassays, known totest the targeted biological activities. Alternatively, bioassays can beused to screen candidates directly. Some of the methods for identifyingand characterizing antibodies are described in detail in the Examples.

CD70 antibodies may be characterized using methods well known in theart. For example, one method is to identify the epitope to which itbinds, or “epitope mapping.” There are many methods known in the art formapping and characterizing the location of epitopes on proteins,including solving the crystal structure of an antibody-antigen complex,competition assays, gene fragment expression assays, and syntheticpeptide-based assays, as described, for example, in Chapter 11 of Harlowand Lane, Using Antibodies, a Laboratory Manual, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1999. In an additionalexample, epitope mapping can be used to determine the sequence to whichan antibody binds. Epitope mapping is commercially available fromvarious sources, for example, Pepscan Systems (Edelhertweg 15, 8219 PHLelystad, The Netherlands). The epitope can be a linear epitope, i.e.,contained in a single stretch of amino acids, or a conformationalepitope formed by a three-dimensional interaction of amino acids thatmay not necessarily be contained in a single stretch. Peptides ofvarying lengths (e.g., at least 4-6 amino acids long) can be isolated orsynthesized (e.g., recombinantly) and used for binding assays with anCD70 or other tumor antigen antibody. In another example, the epitope towhich the CD70 antibody binds can be determined in a systematicscreening by using overlapping peptides derived from the CD70 sequenceand determining binding by the CD70 antibody. According to the genefragment expression assays, the open reading frame encoding CD70 isfragmented either randomly or by specific genetic constructions and thereactivity of the expressed fragments of CD70 with the antibody to betested is determined. The gene fragments may, for example, be producedby PCR and then transcribed and translated into protein in vitro, in thepresence of radioactive amino acids. The binding of the antibody to theradioactively labeled CD70 is then determined by immunoprecipitation andgel electrophoresis. Certain epitopes can also be identified by usinglarge libraries of random peptide sequences displayed on the surface ofphage particles (phage libraries). Alternatively, a defined library ofoverlapping peptide fragments can be tested for binding to the testantibody in simple binding assays. In an additional example, mutagenesisof an antigen binding domain, domain swapping experiments and alaninescanning mutagenesis can be performed to identify residues required,sufficient, or necessary for epitope binding. For example, domainswapping experiments can be performed using a mutant CD70 in whichvarious fragments of the CD70 protein have been replaced (swapped) withsequences from CD70 from another species (e.g., mouse), or a closelyrelated, but antigenically distinct protein. By assessing binding of theantibody to the mutant CD70, the importance of the particular CD70fragment to antibody binding can be assessed. In the case of CD70specific antibody (i.e. antibody that does not bind CD70 wt (wild type)or any other proteins), epitope can be deduced from the sequencealignment of CD70 to CD70 wt.

Yet another method which can be used to characterize an CD70 antibody isto use competition assays with other antibodies known to bind to thesame antigen, i.e., various fragments on CD70, to determine if the CD70antibody binds to the same epitope as other antibodies. Competitionassays are well known to those of skill in the art.

An expression vector can be used to direct expression of an CD70antibody. One skilled in the art is familiar with administration ofexpression vectors to obtain expression of an exogenous protein in vivo.See, e.g., U.S. Pat. Nos. 6,436,908; 6,413,942; and 6,376,471.Administration of expression vectors includes local or systemicadministration, including injection, oral administration, particle gunor catheterized administration, and topical administration. In anotherembodiment, the expression vector is administered directly to thesympathetic trunk or ganglion, or into a coronary artery, atrium,ventrical, or pericardium.

Targeted delivery of therapeutic compositions containing an expressionvector, or subgenomic polynucleotides can also be used.Receptor-mediated DNA delivery techniques are described in, for example,Findeis et al., Trends Biotechnol., 1993, 11:202; Chiou et al., GeneTherapeutics: Methods And Applications Of Direct Gene Transfer, J. A.Wolff, ed., 1994; Wu et al., J. Biol. Chem., 263:621, 1988; Wu et al.,J. Biol. Chem., 269:542, 1994; Zenke et al., Proc. Natl. Acad. Sci. USA,87:3655, 1990; and Wu et al., J. Biol. Chem., 266:338, 1991. Therapeuticcompositions containing a polynucleotide are administered in a range ofabout 100 ng to about 200 mg of DNA for local administration in a genetherapy protocol. Concentration ranges of about 500 ng to about 50 mg,about 1 to about 2 mg, about 5 μg to about 500 μg, and about 20 μg toabout 100 μg of DNA can also be used during a gene therapy protocol. Thetherapeutic polynucleotides and polypeptides can be delivered using genedelivery vehicles. The gene delivery vehicle can be of viral ornon-viral origin (see generally, Jolly, Cancer Gene Therapy, 1:51, 1994;Kimura, Human Gene Therapy, 5:845, 1994; Connelly, Human Gene Therapy,1995, 1:185; and Kaplitt, Nature Genetics, 6:148, 1994). Expression ofsuch coding sequences can be induced using endogenous mammalian orheterologous promoters. Expression of the coding sequence can be eitherconstitutive or regulated.

Viral-based vectors for delivery of a desired polynucleotide andexpression in a desired cell are well known in the art. Exemplaryviral-based vehicles include, but are not limited to, recombinantretroviruses (see, e.g., PCT Publication Nos. WO 90/07936; WO 94/03622;WO 93/25698; WO 93/25234; WO 93/11230; WO 93/10218; WO 91/02805; U.S.Pat. Nos. 5,219,740 and 4,777,127; GB Pat. No. 2,200,651; and EP Pat.No. 0 345 242), alphavirus-based vectors (e.g., Sindbis virus vectors,Semliki forest virus (ATCC VR-67; ATCC VR-1247), Ross River virus (ATCCVR-373; ATCC VR-1246) and Venezuelan equine encephalitis virus (ATCCVR-923; ATCC VR-1250; ATCC VR 1249; ATCC VR-532)), and adeno-associatedvirus (AAV) vectors (see, e.g., PCT Publication Nos. WO 94/12649, WO93/03769; WO 93/19191; WO 94/28938; WO 95/11984 and WO 95/00655).Administration of DNA linked to killed adenovirus as described inCuriel, Hum. Gene Ther., 1992, 3:147 can also be employed.

Non-viral delivery vehicles and methods can also be employed, including,but not limited to, polycationic condensed DNA linked or unlinked tokilled adenovirus alone (see, e.g., Curiel, Hum. Gene Ther., 3:147,1992); ligand-linked DNA (see, e.g., Wu, J. Biol. Chem., 264:16985,1989); eukaryotic cell delivery vehicles cells (see, e.g., U.S. Pat. No.5,814,482; PCT Publication Nos. WO 95/07994; WO 96/17072; WO 95/30763;and WO 97/42338) and nucleic charge neutralization or fusion with cellmembranes. Naked DNA can also be employed. Exemplary naked DNAintroduction methods are described in PCT Publication No. WO 90/11092and U.S. Pat. No. 5,580,859. Liposomes that can act as gene deliveryvehicles are described in U.S. Pat. No. 5,422,120; PCT Publication Nos.WO 95/13796; WO 94/23697; WO 91/14445; and EP 0524968. Additionalapproaches are described in Philip, Mol. Cell Biol., 14:2411, 1994 andin Woffendin, Proc. Natl. Acad. Sci., 91:1581, 1994.

In some embodiments, the invention encompasses compositions, includingpharmaceutical compositions, comprising antibodies described herein ormade by the methods and having the characteristics described herein. Asused herein, compositions comprise one or more antibodies that bind toCD70, or one or more polynucleotides comprising sequences encoding oneor more these antibodies. These compositions may further comprisesuitable excipients, such as pharmaceutically acceptable excipientsincluding buffers, which are well known in the art.

The invention also provides methods of making any of these antibodies.The antibodies of this invention can be made by procedures known in theart. The polypeptides can be produced by proteolytic or otherdegradation of the antibodies, by recombinant methods (i.e., single orfusion polypeptides) as described above or by chemical synthesis.Polypeptides of the antibodies, especially shorter polypeptides up toabout 50 amino acids, are conveniently made by chemical synthesis.Methods of chemical synthesis are known in the art and are commerciallyavailable. For example, an antibody could be produced by an automatedpolypeptide synthesizer employing the solid phase method. See also, U.S.Pat. Nos. 5,807,715; 4,816,567; and 6,331,415.

In another alternative, the antibodies can be made recombinantly usingprocedures that are well known in the art. In one embodiment, apolynucleotide comprises a sequence encoding the heavy chain or thelight chain variable regions of antibody 31H1, 6362, 40E3, 42C3, 45F11,64F9, 72C2, 2F10, 4F11, 10H10, 17G6, 65E11, P02B10, P07D03, P08A02,P08E02, P08F08, P08G02, P12B09, P12F02, P12G07, P13F04, P15D02 orP16C05. The sequence encoding the antibody of interest may be maintainedin a vector in a host cell and the host cell can then be expanded andfrozen for future use. Vectors (including expression vectors) and hostcells are further described herein.

Heteroconjugate antibodies, comprising two covalently joined antibodies,are also within the scope of the invention. Such antibodies have beenused to target immune system cells to unwanted cells (U.S. Pat. No.4,676,980), and for treatment of HIV infection (PCT Publication Nos. WO91/00360 and WO 92/200373; EP 03089). Heteroconjugate antibodies may bemade using any convenient cross-linking methods. Suitable cross-linkingagents and techniques are well known in the art, and are described inU.S. Pat. No. 4,676,980.

Chimeric or hybrid antibodies also may be prepared in vitro using knownmethods of synthetic protein chemistry, including those involvingcross-linking agents. For example, immunotoxins may be constructed usinga disulfide exchange reaction or by forming a thioether bond. Examplesof suitable reagents for this purpose include iminothiolate andmethyl-4-mercaptobutyrim idate.

In the recombinant humanized antibodies, the Fcγ portion can be modifiedto avoid interaction with Fcγ receptor and the complement and immunesystems. The techniques for preparation of such antibodies are describedin WO 99/58572. For example, the constant region may be engineered tomore resemble human constant regions to avoid immune response if theantibody is used in clinical trials and treatments in humans. See, forexample, U.S. Pat. Nos. 5,997,867 and 5,866,692.

The invention encompasses modifications to the antibodies andpolypeptides of the invention including variants shown in Table 5,including functionally equivalent antibodies which do not significantlyaffect their properties and variants which have enhanced or decreasedactivity or affinity. For example, the amino acid sequence may bemutated to obtain an antibody with the desired binding affinity to CD70.Modification of polypeptides is routine practice in the art and need notbe described in detail herein. Examples of modified polypeptides includepolypeptides with conservative substitutions of amino acid residues, oneor more deletions or additions of amino acids which do not significantlydeleteriously change the functional activity, or which mature (enhance)the affinity of the polypeptide for its ligand, or use of chemicalanalogs.

Amino acid sequence insertions include amino- or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includean antibody with an N-terminal methionyl residue or the antibody fusedto an epitope tag. Other insertional variants of the antibody moleculeinclude the fusion to the N- or C-terminus of the antibody of an enzymeor a polypeptide which increases the half-life of the antibody in theblood circulation.

Substitution variants have at least one amino acid residue in theantibody molecule removed and a different residue inserted in its place.The sites of greatest interest for substitutional mutagenesis includethe hypervariable regions, but FR alterations are also contemplated.Conservative substitutions are shown in Table 5 under the heading of“conservative substitutions.” If such substitutions result in a changein biological activity, then more substantial changes, denominated“exemplary substitutions” in Table 5, or as further described below inreference to amino acid classes, may be introduced and the productsscreened. In some embodiments, substitution variants of antibodiesprovided herein have no more than 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5,4, 3, 2, or 1 conservative substitution in the VH or VL region ascompared to the reference parent antibody. In some embodiments, thesubstitutions are not within a CDR of the VH or VL region.

TABLE 5 Amino Acid Substitutions Original Residue (naturally occurringamino Conservative acid) Substitutions Exemplary Substitutions Ala (A)Val Val; Leu; Ile Arg (R) Lys Lys; Gln; Asn Asn (N) Gln Gln; His; Asp,Lys; Arg Asp (D) Glu Glu; Asn Cys (C) Ser Ser; Ala Gln (Q) Asn Asn; GluGlu (E) Asp Asp; Gln Gly (G) Ala Ala His (H) Arg Asn; Gln; Lys; Arg Ile(I) Leu Leu; Val; Met; Ala; Phe; Norleucine Leu (L) Ile Norleucine; Ile;Val; Met; Ala; Phe Lys (K) Arg Arg; Gln; Asn Met (M) Leu Leu; Phe; IlePhe (F) Tyr Leu; Val; Ile; Ala; Tyr Pro (P) Ala Ala Ser (S) Thr Thr Thr(T) Ser Ser Trp (W) Tyr Tyr; Phe Tyr (Y) Phe Trp; Phe; Thr; Ser Val (V)Leu Ile; Leu; Met; Phe; Ala; Norleucine

Substantial modifications in the biological properties of the antibodyare accomplished by selecting substitutions that differ significantly intheir effect on maintaining (a) the structure of the polypeptidebackbone in the area of the substitution, for example, as a sheet orhelical conformation, (b) the charge or hydrophobicity of the moleculeat the target site, or (c) the bulk of the side chain. Naturallyoccurring amino acid residues are divided into groups based on commonside-chain properties:

-   -   (1) Non-polar: Norleucine, Met, Ala, Val, Leu, Ile;    -   (2) Polar without charge: Cys, Ser, Thr, Asn, Gin;    -   (3) Acidic (negatively charged): Asp, Glu;    -   (4) Basic (positively charged): Lys, Arg;    -   (5) Residues that influence chain orientation: Gly, Pro; and    -   (6) Aromatic: Trp, Tyr, Phe, His.

Non-conservative substitutions are made by exchanging a member of one ofthese classes for another class.

Any cysteine residue not involved in maintaining the proper conformationof the antibody also may be substituted, generally with serine, toimprove the oxidative stability of the molecule and prevent aberrantcross-linking. Conversely, cysteine bond(s) may be added to the antibodyto improve its stability, particularly where the antibody is an antibodyfragment such as an Fv fragment.

Amino acid modifications can range from changing or modifying one ormore amino acids to complete redesign of a region, such as the variableregion. Changes in the variable region can alter binding affinity orspecificity. In some embodiments, no more than one to five conservativeamino acid substitutions are made within a CDR domain. In otherembodiments, no more than one to three conservative amino acidsubstitutions are made within a CDR domain. In still other embodiments,the CDR domain is CDR H3 or CDR L3.

Modifications also include glycosylated and nonglycosylatedpolypeptides, as well as polypeptides with other post-translationalmodifications, such as, for example, glycosylation with differentsugars, acetylation, and phosphorylation. Antibodies are glycosylated atconserved positions in their constant regions (Jefferis and Lund, Chem.Immunol. 65:111-128, 1997; Wright and Morrison, TibTECH 15:26-32, 1997).The oligosaccharide side chains of the immunoglobulins affect theprotein's function (Boyd et al., Mol. Immunol. 32:1311-1318, 1996;Wittwe and Howard, Biochem. 29:4175-4180, 1990) and the intramolecularinteraction between portions of the glycoprotein, which can affect theconformation and presented three-dimensional surface of the glycoprotein(Jefferis and Lund, supra; Wyss and Wagner, Current Opin. Biotech.7:409-416, 1996). Oligosaccharides may also serve to target a givenglycoprotein to certain molecules based upon specific recognitionstructures. Glycosylation of antibodies has also been reported to affectantibody-dependent cellular cytotoxicity (ADCC). In particular, CHOcells with tetracycline-regulated expression ofβ(1,4)-N-acetylglucosaminyltransferase III (GnTIII), aglycosyltransferase catalyzing formation of bisecting GlcNAc, wasreported to have improved ADCC activity (Umana et al., Mature Biotech.17:176-180, 1999).

Glycosylation of antibodies is typically either N-linked or O-linked.N-linked refers to the attachment of the carbohydrate moiety to the sidechain of an asparagine residue. The tripeptide sequencesasparagine-X-serine, asparagine-X-threonine, and asparagine-X-cysteine,where X is any amino acid except proline, are the recognition sequencesfor enzymatic attachment of the carbohydrate moiety to the asparagineside chain. Thus, the presence of either of these tripeptide sequencesin a polypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, mostcommonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used.

Addition of glycosylation sites to the antibody is convenientlyaccomplished by altering the amino acid sequence such that it containsone or more of the above-described tripeptide sequences (for N-linkedglycosylation sites). The alteration may also be made by the additionof, or substitution by, one or more serine or threonine residues to thesequence of the original antibody (for O-linked glycosylation sites).

The glycosylation pattern of antibodies may also be altered withoutaltering the underlying nucleotide sequence. Glycosylation largelydepends on the host cell used to express the antibody. Since the celltype used for expression of recombinant glycoproteins, e.g. antibodies,as potential therapeutics is rarely the native cell, variations in theglycosylation pattern of the antibodies can be expected (see, e.g. Hseet al., J. Biol. Chem. 272:9062-9070, 1997).

In addition to the choice of host cells, factors that affectglycosylation during recombinant production of antibodies include growthmode, media formulation, culture density, oxygenation, pH, purificationschemes and the like. Various methods have been proposed to alter theglycosylation pattern achieved in a particular host organism includingintroducing or overexpressing certain enzymes involved inoligosaccharide production (U.S. Pat. Nos. 5,047,335; 5,510,261 and5,278,299). Glycosylation, or certain types of glycosylation, can beenzymatically removed from the glycoprotein, for example, usingendoglycosidase H (Endo H), N-glycosidase F, endoglycosidase F1,endoglycosidase F2, endoglycosidase F3. In addition, the recombinanthost cell can be genetically engineered to be defective in processingcertain types of polysaccharides. These and similar techniques are wellknown in the art.

Other methods of modification include using coupling techniques known inthe art, including, but not limited to, enzymatic means, oxidativesubstitution and chelation.

Modifications can be used, for example, for attachment of labels forimmunoassay. Modified polypeptides are made using established proceduresin the art and can be screened using standard assays known in the art,some of which are described below and in the Examples.

Other antibody modifications include antibodies that have been modifiedas described in PCT Publication No. WO 99/58572. These antibodiescomprise, in addition to a binding domain directed at the targetmolecule, an effector domain having an amino acid sequence substantiallyhomologous to all or part of a constant region of a human immunoglobulinheavy chain. These antibodies are capable of binding the target moleculewithout triggering significant complement dependent lysis, orcell-mediated destruction of the target. In some embodiments, theeffector domain is capable of specifically binding FcRn or FcγRIIb.These are typically based on chimeric domains derived from two or morehuman immunoglobulin heavy chain CH2 domains. Antibodies modified inthis manner are particularly suitable for use in chronic antibodytherapy, to avoid inflammatory and other adverse reactions toconventional antibody therapy.

The invention includes affinity matured embodiments. For example,affinity matured antibodies can be produced by procedures known in theart (Marks et al., Bio/Technology, 10:779-783, 1992; Barbas et al., ProcNat. Acad. Sci, USA 91:3809-3813, 1994; Schier et al., Gene,169:147-155, 1995; Yelton et al., J. Immunol., 155:1994-2004, 1995;Jackson et al., J. Immunol., 154(7):3310-9, 1995, Hawkins et al., J.Mol. Biol., 226:889-896, 1992; and PCT Publication No. WO2004/058184).

The following methods may be used for adjusting the affinity of anantibody and for characterizing a CDR. One way of characterizing a CDRof an antibody or altering (such as improving) the binding affinity of apolypeptide, such as an antibody, termed “library scanning mutagenesis”.Generally, library scanning mutagenesis works as follows. One or moreamino acid positions in the CDR are replaced with two or more (such as3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20)amino acids using art recognized methods. This generates small librariesof clones (in some embodiments, one for every amino acid position thatis analyzed), each with a complexity of two or more members (if two ormore amino acids are substituted at every position). Generally, thelibrary also includes a clone comprising the native (unsubstituted)amino acid. A small number of clones, e.g., about 20-80 clones(depending on the complexity of the library), from each library arescreened for binding affinity to the target polypeptide (or otherbinding target), and candidates with increased, the same, decreased, orno binding are identified. Methods for determining binding affinity arewell-known in the art. Binding affinity may be determined using Biacore™surface plasmon resonance analysis, which detects differences in bindingaffinity of about 2-fold or greater. Biacore™ is particularly usefulwhen the starting antibody already binds with a relatively highaffinity, for example a Ko of about 10 nM or lower. Screening usingBiacore™ surface plasmon resonance is described in the Examples, herein.

Binding affinity may be determined using Kinexa Biocensor, scintillationproximity assays, ELISA, ORIGEN immunoassay (IGEN), fluorescencequenching, fluorescence transfer, or yeast display. Binding affinity mayalso be screened using a suitable bioassay.

In some embodiments, every amino acid position in a CDR is replaced (insome embodiments, one at a time) with all 20 natural amino acids usingart recognized mutagenesis methods (some of which are described herein).This generates small libraries of clones (in some embodiments, one forevery amino acid position that is analyzed), each with a complexity of20 members (if all 20 amino acids are substituted at every position).

In some embodiments, the library to be screened comprises substitutionsin two or more positions, which may be in the same CDR or in two or moreCDRs. Thus, the library may comprise substitutions in two or morepositions in one CDR. The library may comprise substitution in two ormore positions in two or more CDRs. The library may comprisesubstitution in 3, 4, 5, or more positions, said positions found in two,three, four, five or six CDRs. The substitution may be prepared usinglow redundancy codons. See, e.g., Table 2 of Balint et al., Gene137(1):109-18, 1993.

The CDR may be CDRH3 or CDRL3. The CDR may be one or more of CDRL1,CDRL2, CDRL3, CDRH1, CDRH2, or CDRH3. The CDR may be a Kabat CDR, aChothia CDR, or an extended CDR.

Candidates with improved binding may be sequenced, thereby identifying aCDR substitution mutant which results in improved affinity (also termedan “improved” substitution). Candidates that bind may also be sequenced,thereby identifying a CDR substitution which retains binding.

Multiple rounds of screening may be conducted. For example, candidates(each comprising an amino acid substitution at one or more position ofone or more CDR) with improved binding are also useful for the design ofa second library containing at least the original and substituted aminoacid at each improved CDR position (i.e., amino acid position in the CDRat which a substitution mutant showed improved binding). Preparation,and screening or selection of this library is discussed further below.

Library scanning mutagenesis also provides a means for characterizing aCDR, in so far as the frequency of clones with improved binding, thesame binding, decreased binding or no binding also provide informationrelating to the importance of each amino acid position for the stabilityof the antibody-antigen complex. For example, if a position of the CDRretains binding when changed to all 20 amino acids, that position isidentified as a position that is unlikely to be required for antigenbinding. Conversely, if a position of CDR retains binding in only asmall percentage of substitutions, that position is identified as aposition that is important to CDR function. Thus, the library scanningmutagenesis methods generate information regarding positions in the CDRsthat can be changed to many different amino acids (including all 20amino acids), and positions in the CDRs which cannot be changed or whichcan only be changed to a few amino acids.

Candidates with improved affinity may be combined in a second library,which includes the improved amino acid, the original amino acid at thatposition, and may further include additional substitutions at thatposition, depending on the complexity of the library that is desired, orpermitted using the desired screening or selection method. In addition,if desired, adjacent amino acid position can be randomized to at leasttwo or more amino acids. Randomization of adjacent amino acids maypermit additional conformational flexibility in the mutant CDR, whichmay in turn, permit or facilitate the introduction of a larger number ofimproving mutations. The library may also comprise substitution atpositions that did not show improved affinity in the first round ofscreening.

The second library is screened or selected for library members withimproved or altered binding affinity using any method known in the art,including screening using Biacore™ surface plasmon resonance analysis,and selection using any method known in the art for selection, includingphage display, yeast display, and ribosome display.

The invention also encompasses fusion proteins comprising one or morefragments or regions from the antibodies of this invention. In oneembodiment, a fusion polypeptide is provided that comprises at least 10contiguous amino acids of the variable light chain region shown in SEQID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33,35, 37, 39, 41, 43, 45 or 47, or at least 10 amino acids of the variableheavy chain region shown in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18,20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46 or 48. In otherembodiments, a fusion polypeptide is provided that comprises at leastabout 10, at least about 15, at least about 20, at least about 25, or atleast about 30 contiguous amino acids of the variable light chain regionor at least about 10, at least about 15, at least about 20, at leastabout 25, or at least about 30 contiguous amino acids of the variableheavy chain region. In another embodiment, the fusion polypeptidecomprises one or more CDR(s). In still other embodiments, the fusionpolypeptide comprises CDR H3 (VH CDR3) or CDR L3 (VL CDR3). For purposesof this invention, a fusion protein contains one or more antibodies andanother amino acid sequence to which it is not attached in the nativemolecule, for example, a heterologous sequence or a homologous sequencefrom another region. Exemplary heterologous sequences include, but arenot limited to a “tag” such as a FLAG tag or a 6His tag (SEQ ID NO:531). Tags are well known in the art.

A fusion polypeptide can be created by methods known in the art, forexample, synthetically or recombinantly. Typically, the fusion proteinsof this invention are made by preparing an expressing a polynucleotideencoding them using recombinant methods described herein, although theymay also be prepared by other means known in the art, including, forexample, chemical synthesis.

This invention also provides compositions comprising antibodiesconjugated (for example, linked) to an agent that facilitate coupling toa solid support (such as biotin or avidin). For simplicity, referencewill be made generally to antibodies with the understanding that thesemethods apply to any of the CD70 antibody embodiments described herein.Conjugation generally refers to linking these components as describedherein. The linking (which is generally fixing these components inproximate association at least for administration) can be achieved inany number of ways. For example, a direct reaction between an agent andan antibody is possible when each possesses a substituent capable ofreacting with the other. For example, a nucleophilic group, such as anamino or sulfhydryl group, on one may be capable of reacting with acarbonyl-containing group, such as an anhydride or an acid halide, orwith an alkyl group containing a good leaving group (e.g., a halide) onthe other.

The invention also provides isolated polynucleotides encoding theantibodies of the invention, and vectors and host cells comprising thepolynucleotide.

Accordingly, the invention provides polynucleotides (or compositions,including pharmaceutical compositions), comprising polynucleotidesencoding any of the following: 31H1, 6362, 40E3, 42C3, 45F11, 64F9,72C2, 2F10, 4F11, 10H10, 17G6, 65E11, P02B10, P07D03, P08A02, P08E02,P08F08, P08G02, P12B09, P12F02, P12G07, P13F04, P15D02 or P16C05, or anyfragment or part thereof having the ability to bind CD70.

In another aspect, the invention provides polynucleotides encoding anyof the antibodies (including antibody fragments) and polypeptidesdescribed herein, such as antibodies and polypeptides having impairedeffector function. Polynucleotides can be made and expressed byprocedures known in the art.

In another aspect, the invention provides compositions (such as apharmaceutical compositions) comprising any of the polynucleotides ofthe invention. In some embodiments, the composition comprises anexpression vector comprising a polynucleotide encoding any of theantibodies described herein.

Expression vectors, and administration of polynucleotide compositionsare further described herein.

In another aspect, the invention provides a method of making any of thepolynucleotides described herein.

Polynucleotides complementary to any such sequences are also encompassedby the present invention. Polynucleotides may be single-stranded (codingor antisense) or double-stranded, and may be DNA (genomic, cDNA orsynthetic) or RNA molecules. RNA molecules include HnRNA molecules,which contain introns and correspond to a DNA molecule in a one-to-onemanner, and mRNA molecules, which do not contain introns. Additionalcoding or non-coding sequences may, but need not, be present within apolynucleotide of the present invention, and a polynucleotide may, butneed not, be linked to other molecules or support materials.

Polynucleotides may comprise a native sequence (i.e., an endogenoussequence that encodes an antibody or a portion thereof) or may comprisea variant of such a sequence. Polynucleotide variants contain one ormore substitutions, additions, deletions or insertions such that theimmunoreactivity of the encoded polypeptide is not diminished, relativeto a native immunoreactive molecule. The effect on the immunoreactivityof the encoded polypeptide may generally be assessed as describedherein. Variants preferably exhibit at least about 70% identity, morepreferably, at least about 80% identity, yet more preferably, at leastabout 90% identity, and most preferably, at least about 95% identity toa polynucleotide sequence that encodes a native antibody or a portionthereof.

Two polynucleotide or polypeptide sequences are said to be “identical”if the sequence of nucleotides or amino acids in the two sequences isthe same when aligned for maximum correspondence as described below.Comparisons between two sequences are typically performed by comparingthe sequences over a comparison window to identify and compare localregions of sequence similarity. A “comparison window” as used herein,refers to a segment of at least about 20 contiguous positions, usually30 to about 75, or 40 to about 50, in which a sequence may be comparedto a reference sequence of the same number of contiguous positions afterthe two sequences are optimally aligned.

Optimal alignment of sequences for comparison may be conducted using theMegalign program in the Lasergene suite of bioinformatics software(DNASTAR, Inc., Madison, Wis.), using default parameters. This programembodies several alignment schemes described in the followingreferences: Dayhoff, M. O., 1978, A model of evolutionary change inproteins—Matrices for detecting distant relationships. In Dayhoff, M. O.(ed.) Atlas of Protein Sequence and Structure, National BiomedicalResearch Foundation, Washington D.C. Vol. 5, Suppl. 3, pp. 345-358; HeinJ., 1990, Unified Approach to Alignment and Phylogenes pp. 626-645Methods in Enzymology vol. 183, Academic Press, Inc., San Diego, Calif.;Higgins, D. G. and Sharp, P. M., 1989, CABIOS 5:151-153; Myers, E. W.and Muller W., 1988, CABIOS 4:11-17; Robinson, E. D., 1971, Comb. Theor.11:105; Santou, N., Nes, M., 1987, Mol. Biol. Evol. 4:406-425; Sneath,P. H. A. and Sokal, R. R., 1973, Numerical Taxonomy the Principles andPractice of Numerical Taxonomy, Freeman Press, San Francisco, Calif.;Wilbur, W. J. and Lipman, D. J., 1983, Proc. Natl. Acad. Sci. USA80:726-730.

Preferably, the “percentage of sequence identity” is determined bycomparing two optimally aligned sequences over a window of comparison ofat least 20 positions, wherein the portion of the polynucleotide orpolypeptide sequence in the comparison window may comprise additions ordeletions (i.e., gaps) of 20 percent or less, usually 5 to 15 percent,or 10 to 12 percent, as compared to the reference sequences (which doesnot comprise additions or deletions) for optimal alignment of the twosequences. The percentage is calculated by determining the number ofpositions at which the identical nucleic acid bases or amino acidresidue occurs in both sequences to yield the number of matchedpositions, dividing the number of matched positions by the total numberof positions in the reference sequence (i.e. the window size) andmultiplying the results by 100 to yield the percentage of sequenceidentity.

Variants may also, or alternatively, be substantially homologous to anative gene, or a portion or complement thereof. Such polynucleotidevariants are capable of hybridizing under moderately stringentconditions to a naturally occurring DNA sequence encoding a nativeantibody (or a complementary sequence).

Suitable “moderately stringent conditions” include prewashing in asolution of 5×SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0); hybridizing at 50°C.−65° C., 5×SSC, overnight; followed by washing twice at 65° C. for 20minutes with each of 2×, 0.5× and 0.2×SSC containing 0.1% SDS.

As used herein, “highly stringent conditions” or “high stringencyconditions” are those that: (1) employ low ionic strength and hightemperature for washing, for example 0.015 M sodium chloride/0.0015 Msodium citrate/0.1% sodium dodecyl sulfate at 50° C.; (2) employ duringhybridization a denaturing agent, such as formamide, for example, 50%(v/v) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1%polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mMsodium chloride, 75 mM sodium citrate at 42° C.; or (3) employ 50%formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodiumphosphate (pH 6.8), 0.1% sodium pyrophosphate, 5×Denhardt's solution,sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10% dextran sulfateat 42° C., with washes at 42° C. in 0.2×SSC (sodium chloride/sodiumcitrate) and 50% formamide at 55° C., followed by a high-stringency washconsisting of 0.1×SSC containing EDTA at 55° C. The skilled artisan willrecognize how to adjust the temperature, ionic strength, etc. asnecessary to accommodate factors such as probe length and the like.

It will be appreciated by those of ordinary skill in the art that, as aresult of the degeneracy of the genetic code, there are many nucleotidesequences that encode a polypeptide as described herein. Some of thesepolynucleotides bear minimal homology to the nucleotide sequence of anynative gene. Nonetheless, polynucleotides that vary due to differencesin codon usage are specifically contemplated by the present invention.Further, alleles of the genes comprising the polynucleotide sequencesprovided herein are within the scope of the present invention. Allelesare endogenous genes that are altered as a result of one or moremutations, such as deletions, additions or substitutions of nucleotides.The resulting mRNA and protein may, but need not, have an alteredstructure or function. Alleles may be identified using standardtechniques (such as hybridization, amplification or database sequencecomparison).

The polynucleotides of this invention can be obtained using chemicalsynthesis, recombinant methods, or PCR. Methods of chemicalpolynucleotide synthesis are well known in the art and need not bedescribed in detail herein. One of skill in the art can use thesequences provided herein and a commercial DNA synthesizer to produce adesired DNA sequence.

For preparing polynucleotides using recombinant methods, apolynucleotide comprising a desired sequence can be inserted into asuitable vector, and the vector in turn can be introduced into asuitable host cell for replication and amplification, as furtherdiscussed herein. Polynucleotides may be inserted into host cells by anymeans known in the art. Cells are transformed by introducing anexogenous polynucleotide by direct uptake, endocytosis, transfection,F-mating or electroporation. Once introduced, the exogenouspolynucleotide can be maintained within the cell as a non-integratedvector (such as a plasmid) or integrated into the host cell genome. Thepolynucleotide so amplified can be isolated from the host cell bymethods well known within the art. See, e.g., Sambrook et al., 1989.

Alternatively, PCR allows reproduction of DNA sequences. PCR technologyis well known in the art and is described in U.S. Pat. Nos. 4,683,195,4,800,159, 4,754,065 and 4,683,202, as well as PCR: The Polymerase ChainReaction, Mullis et al. eds., Birkauswer Press, Boston, 1994.

RNA can be obtained by using the isolated DNA in an appropriate vectorand inserting it into a suitable host cell. When the cell replicates andthe DNA is transcribed into RNA, the RNA can then be isolated usingmethods well known to those of skill in the art, as set forth inSambrook et al., 1989, supra, for example.

Suitable cloning vectors may be constructed according to standardtechniques, or may be selected from a large number of cloning vectorsavailable in the art. While the cloning vector selected may varyaccording to the host cell intended to be used, useful cloning vectorswill generally have the ability to self-replicate, may possess a singletarget for a particular restriction endonuclease, or may carry genes fora marker that can be used in selecting clones containing the vector.Suitable examples include plasmids and bacterial viruses, e.g., pUC18,pUC19, Bluescript (e.g., pBS SK+) and its derivatives, mp18, mp19,pBR322, pMB9, ColE1, pCR1, RP4, phage DNAs, and shuttle vectors such aspSA3 and pAT28. These and many other cloning vectors are available fromcommercial vendors such as BioRad, Strategene, and Invitrogen.

Expression vectors generally are replicable polynucleotide constructsthat contain a polynucleotide according to the invention. It is impliedthat an expression vector must be replicable in the host cells either asepisomes or as an integral part of the chromosomal DNA. Suitableexpression vectors include but are not limited to plasmids, viralvectors, including adenoviruses, adeno-associated viruses, retroviruses,cosmids, and expression vector(s) disclosed in PCT Publication No. WO87/04462. Vector components may generally include, but are not limitedto, one or more of the following: a signal sequence; an origin ofreplication; one or more marker genes; suitable transcriptionalcontrolling elements (such as promoters, enhancers and terminator). Forexpression (i.e., translation), one or more translational controllingelements are also usually required, such as ribosome binding sites,translation initiation sites, and stop codons.

The vectors containing the polynucleotides of interest can be introducedinto the host cell by any of a number of appropriate means, includingelectroporation, transfection employing calcium chloride, rubidiumchloride, calcium phosphate, DEAE-dextran, or other substances;microprojectile bombardment; lipofection; and infection (e.g., where thevector is an infectious agent such as vaccinia virus). The choice ofintroducing vectors or polynucleotides will often depend on features ofthe host cell.

The invention also provides host cells comprising any of thepolynucleotides described herein. Any host cells capable ofover-expressing heterologous DNAs can be used for the purpose ofisolating the genes encoding the antibody, polypeptide or protein ofinterest. Non-limiting examples of mammalian host cells include but notlimited to COS, HeLa, and CHO cells. See also PCT Publication No. WO87/04462. Suitable non-mammalian host cells include prokaryotes (such asE. coli or B. subtilis) and yeast (such as S. cerevisae, S. pombe; or K.lactis). Preferably, the host cells express the cDNAs at a level ofabout 5 fold higher, more preferably, 10 fold higher, even morepreferably, 20 fold higher than that of the corresponding endogenousantibody or protein of interest, if present, in the host cells.Screening the host cells for a specific binding to CD70 is effected byan immunoassay or FACS. A cell overexpressing the antibody or protein ofinterest can be identified.

Methods of Using the CD70 Antibodies

The antibodies of the present invention are useful in variousapplications including, but are not limited to, therapeutic treatmentmethods and diagnostic treatment methods.

The antibodies (e.g., monospecific and bispecific) obtained by themethods described above can be used as a medicament. In someembodiments, such a medicament can be used for treating cancer. In someembodiments, the cancer is a cancer of hematopoietic origin, such as alymphoma or leukemia. In some embodiments, the cancer is Renal CellCarcinoma, Glioblastoma, glioma such as low grade glioma, Non-Hodgkin'sLymphoma (NHL), Hodgkin's Disease (HD), Waldenstrom's macroglobulinemia,Acute Myeloid Leukemia, Multiple Myeloma, diffuse large-cell lymphoma,follicular lymphoma or Non-Small Cell Lung Cancer

In some embodiments, provided is a method of inhibiting tumor growth orprogression in a subject who has malignant cells expressing CD70,comprising administering to the subject in need thereof an effectiveamount of a composition comprising the CD70 antibodies (e.g., CD70-CD3bispecific antibodies) as described herein. In other embodiments,provided is a method of inhibiting metastasis of cells expressing CD70in a subject, comprising administering to the subject in need thereof aneffective amount of a composition comprising the CD70 antibodies (e.g.,CD70-CD3 bispecific antibodies) as described herein. In otherembodiments, provided is a method of inducing tumor regression inmalignant cells in a subject, comprising administering to the subject inneed thereof an effective amount of a composition comprising the CD70antibodies (e.g., CD70-CD3 bispecific antibodies) as described herein.

In some embodiments, the antibody (e.g., CD70-CD3 bispecific antibody)according to the invention can be used in the manufacture of amedicament for treatment of a cancer in a patient in need thereof.

In some embodiments, the treatment can be in combination with one ormore therapies against cancer selected from the group of antibodiestherapy, chemotherapy, cytokines therapy, targeted therapy, vaccinetherapy, dendritic cell therapy, gene therapy, hormone therapy, surgicalresection, laser light therapy and radiation therapy.

For example, in some embodiments, the CD70 antibodies (e.g., CD70-CD3bispecific antibodies) of the present invention are administered to apatient in conjunction with (e.g., before, simultaneously or following)treatment with small molecule Tyrosine Kinase Inhibitors (TKIs) such assunitinib and pazopanib that target Vascular Endothelial Growth Factor(VEGF) receptor, monoclonal antibody targeting VEGF such as bevacizumab,mammalian target of Rapamycin (mTOR) inhibitor temsirolimus, as well ashigh dose IL-2. In some embodiments, the CD70 antibodies (e.g., CD70-CD3bispecific antibodies) of the present invention are administered to apatient in conjunction with one or more of the following: an anti-PD-1antibody (e.g., nivolumab, pembrolizumab, or PF-06801591), an anti-PD-L1antibody (e.g., avelumab, atezolizumab, or durvalumab) an anti-OX40antibody (e.g., PF-04518600), an anti-4-1 BB antibody (e.g.,PF-05082566), an anti-MCSF antibody (e.g., PD-0360324), an anti-GITRantibody, or an anti-TIGIT antibody.

The administration of the antibodies (e.g., monospecific or bispecific)according to the invention may be carried out in any convenient manner,including by aerosol inhalation, injection, ingestion, transfusion,implantation or transplantation. The compositions described herein maybe administered to a patient subcutaneously, intradermally,intratumorally, intracranially, intranodally, intramedullary,intramuscularly, by intravenous or intralymphatic injection, orintraperitoneally. In one embodiment, the antibody compositions of theinvention are preferably administered by intravenous injection.

In some embodiments, the administration of the antibodies (e.g.,monospecific or bispecific) can comprise administration of, for example,about 0.01 to about 20 mg per kg body weight including all integervalues of mg per kg within those ranges. In some embodiments, theadministration of the antibodies can comprise administration of about0.1 to 10 mg per kg body weight including all integer values of mg perkg within those ranges. The antibody can be administrated in one or moredoses. In some embodiments, said effective amount of the antibody can beadministrated as a single dose. In some embodiments, said effectiveamount of antibodies can be administrated as more than one dose over aperiod time. Timing of administration is within the judgment of managingphysician and depends on the clinical condition of the patient. Whileindividual needs vary, determination of optimal ranges of effectiveamounts of a given antibody (e.g., monospecific or bispecific) for aparticular disease or conditions within the skill of the art. Aneffective amount means an amount which provides a therapeutic orprophylactic benefit. The dosage administrated will be dependent uponthe age, health and weight of the recipient, kind of concurrenttreatment, if any, frequency of treatment and the nature of the effectdesired. In some embodiments, an effective amount of heteromultimericantibody or composition comprising those antibodies are administratedparenterally. In some embodiments, administration can be an intravenousadministration. In some embodiments, administration can be directly doneby injection within a tumor.

In some embodiments, anti-CD70 antibodies provided herein may be usedfor diagnostic purposes, such in assays to identify CD70 protein insamples (e.g. in immunohistochemistry assays) or in patients.

Compositions

In one aspect, the present invention provides a pharmaceuticalcomposition comprising an antibody (e.g., monospecific or bispecific) ofthe invention or portion thereof as described above in apharmaceutically acceptable carrier. In certain embodiments, thepolypeptides of the invention may be present in a neutral form(including zwitter ionic forms) or as a positively or negatively-chargedspecies. In some embodiments, the polypeptides may be complexed with acounterion to form a “pharmaceutically acceptable salt,” which refers toa complex comprising one or more polypeptides and one or morecounterions, where the counterions are derived from pharmaceuticallyacceptable inorganic and organic acids and bases.

The antibody (e.g., monospecific or bispecific) or portions thereof, maybe administered alone or in combination with one or more otherpolypeptides of the invention or in combination with one or more otherdrugs (or as any combination thereof). The pharmaceutical compositions,methods and uses of the invention thus also encompass embodiments ofcombinations (co-administration) with other active agents, as detailedbelow.

As used herein, the terms “co-administration,” “co-administered” and “incombination with,” referring to the antibodies of the invention and oneor more other therapeutic agents, is intended to mean, and does refer toand include the following: (i) simultaneous administration of suchcombination of an antibody disclosed herein and therapeutic agent(s) toa patient in need of treatment, when such components are formulatedtogether into a single dosage form which releases said components atsubstantially the same time to said patient; (ii) substantiallysimultaneous administration of such combination of an antibody disclosedherein and therapeutic agent(s) to a patient in need of treatment, whensuch components are formulated apart from each other into separatedosage forms which are taken at substantially the same time by saidpatient, whereupon said components are released at substantially thesame time to said patient; (iii) sequential administration of suchcombination of an antibody disclosed herein and therapeutic agent(s) toa patient in need of treatment, when such components are formulatedapart from each other into separate dosage forms which are taken atconsecutive times by said patient with a significant time intervalbetween each administration, whereupon said components are released atsubstantially different times to said patient; and (iv) sequentialadministration of such combination of an antibody disclosed herein andtherapeutic agent(s) to a patient in need of treatment, when suchcomponents are formulated together into a single dosage form whichreleases said components in a controlled manner whereupon they areconcurrently, consecutively, or overlappingly released at the same ordifferent times to said patient, where each part may be administered byeither the same or a different route.

Generally, the antibody (e.g., monospecific or bispecific) disclosedherein or portions thereof are suitable to be administered as aformulation in association with one or more pharmaceutically acceptableexcipient(s). The term ‘excipient’ is used herein to describe anyingredient other than the compound(s) of the invention. The choice ofexcipient(s) will to a large extent depend on factors such as theparticular mode of administration, the effect of the excipient onsolubility and stability, and the nature of the dosage form. As usedherein, “pharmaceutically acceptable excipient” includes any and allsolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like that arephysiologically compatible. Some examples of pharmaceutically acceptableexcipients are water, saline, phosphate buffered saline, dextrose,glycerol, ethanol and the like, as well as combinations thereof. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride inthe composition. Additional examples of pharmaceutically acceptablesubstances are wetting agents or minor amounts of auxiliary substancessuch as wetting or emulsifying agents, preservatives or buffers, whichenhance the shelf life or effectiveness of the antibody.

Pharmaceutical compositions of the present invention and methods fortheir preparation will be readily apparent to those skilled in the art.Such compositions and methods for their preparation may be found, forexample, in Remington's Pharmaceutical Sciences, 19th Edition (MackPublishing Company, 1995). Pharmaceutical compositions are preferablymanufactured under GMP conditions.

A pharmaceutical composition of the invention may be prepared, packaged,or sold in bulk, as a single unit dose, or as a plurality of single unitdoses. As used herein, a “unit dose” is discrete amount of thepharmaceutical composition comprising a predetermined amount of theactive ingredient. The amount of the active ingredient is generallyequal to the dosage of the active ingredient which would be administeredto a subject or a convenient fraction of such a dosage such as, forexample, one-half or one-third of such a dosage. Any method foradministering peptides, proteins or antibodies accepted in the art maysuitably be employed for the heterodimeric proteins and portions thereofdisclosed herein.

The pharmaceutical compositions of the invention are typically suitablefor parenteral administration. As used herein, “parenteraladministration” of a pharmaceutical composition includes any route ofadministration characterized by physical breaching of a tissue of asubject and administration of the pharmaceutical composition through thebreach in the tissue, thus generally resulting in the directadministration into the blood stream, into muscle, or into an internalorgan. Parenteral administration thus includes, but is not limited to,administration of a pharmaceutical composition by injection of thecomposition, by application of the composition through a surgicalincision, by application of the composition through a tissue-penetratingnon-surgical wound, and the like. In particular, parenteraladministration is contemplated to include, but is not limited to,subcutaneous, intraperitoneal, intramuscular, intrasternal, intravenous,intraarterial, intrathecal, intraventricular, intraurethral,intracranial, intrasynovial injection or infusions; and kidney dialyticinfusion techniques. Preferred embodiments include the intravenous andthe subcutaneous routes.

Formulations of a pharmaceutical composition suitable for parenteraladministration typically generally comprise the active ingredientcombined with a pharmaceutically acceptable carrier, such as sterilewater or sterile isotonic saline. Such formulations may be prepared,packaged, or sold in a form suitable for bolus administration or forcontinuous administration. Injectable formulations may be prepared,packaged, or sold in unit dosage form, such as in ampoules or in multidose containers containing a preservative. Formulations for parenteraladministration include, but are not limited to, suspensions, solutions,emulsions in oily or aqueous vehicles, pastes, and the like. Suchformulations may further comprise one or more additional ingredientsincluding, but not limited to, suspending, stabilizing, or dispersingagents. In one embodiment of a formulation for parenteraladministration, the active ingredient is provided in dry (i.e. powder orgranular) form for reconstitution with a suitable vehicle (e.g. sterilepyrogen free water) prior to parenteral administration of thereconstituted composition. Parenteral formulations also include aqueoussolutions which may contain excipients such as salts, carbohydrates andbuffering agents (preferably to a pH of from 3 to 9), but, for someapplications, they may be more suitably formulated as a sterilenon-aqueous solution or as a dried form to be used in conjunction with asuitable vehicle such as sterile, pyrogen-free water. Exemplaryparenteral administration forms include solutions or suspensions insterile aqueous solutions, for example, aqueous propylene glycol ordextrose solutions. Such dosage forms can be suitably buffered, ifdesired. Other parentally-administrable formulations which are usefulinclude those which comprise the active ingredient in microcrystallineform, or in a liposomal preparation. Formulations for parenteraladministration may be formulated to be immediate or modified release.Modified release formulations include controlled, delayed, sustained,pulsed, targeted and programmed release formulations. For example, inone aspect, sterile injectable solutions can be prepared byincorporating the heterodimeric protein, e.g., bispecific antibody, inthe required amount in an appropriate solvent with one or a combinationof ingredients enumerated above, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating theactive compound into a sterile vehicle that contains a basic dispersionmedium and the required other ingredients from those enumerated above.In the case of sterile powders for the preparation of sterile injectablesolutions, the preferred methods of preparation are vacuum drying andfreeze drying that yields a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile filteredsolution thereof. The proper fluidity of a solution can be maintained,for example, by the use of a coating such as lecithin, by themaintenance of the required particle size in the case of dispersion andby the use of surfactants. Prolonged absorption of injectablecompositions can be brought about by including in the composition anagent that delays absorption, for example, monostearate salts andgelatin.

Dosage regimens may be adjusted to provide the optimum desired response.For example, a single bolus may be administered, several divided dosesmay be administered over time or the dose may be proportionally reducedor increased as indicated by the exigencies of the therapeuticsituation. It is especially advantageous to formulate parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form, as used herein, refers tophysically discrete units suited as unitary dosages for thepatients/subjects to be treated; each unit containing a predeterminedquantity of active compound calculated to produce the desiredtherapeutic effect in association with the required pharmaceuticalcarrier. The specification for the dosage unit forms of the inventionare generally dictated by and directly dependent on (a) the uniquecharacteristics of the chemotherapeutic agent and the particulartherapeutic or prophylactic effect to be achieved, and (b) thelimitations inherent in the art of compounding such an active compoundfor the treatment of sensitivity in individuals.

Thus, the skilled artisan would appreciate, based upon the disclosureprovided herein, that the dose and dosing regimen is adjusted inaccordance with methods well-known in the therapeutic arts. That is, themaximum tolerable dose can be readily established, and the effectiveamount providing a detectable therapeutic benefit to a patient may alsobe determined, as can the temporal requirements for administering eachagent to provide a detectable therapeutic benefit to the patient.Accordingly, while certain dose and administration regimens areexemplified herein, these examples in no way limit the dose andadministration regimen that may be provided to a patient in practicingthe present invention.

It is to be noted that dosage values may vary with the type and severityof the condition to be alleviated, and may include single or multipledoses. It is to be further understood that for any particular subject,specific dosage regimens should be adjusted over time according to theindividual need and the professional judgment of the personadministering or supervising the administration of the compositions, andthat dosage ranges set forth herein are exemplary only and are notintended to limit the scope or practice of the claimed composition.Further, the dosage regimen with the compositions of this invention maybe based on a variety of factors, including the type of disease, theage, weight, sex, medical condition of the patient, the severity of thecondition, the route of administration, and the particular antibodyemployed. Thus, the dosage regimen can vary widely, but can bedetermined routinely using standard methods. For example, doses may beadjusted based on pharmacokinetic or pharmacodynamic parameters, whichmay include clinical effects such as toxic effects or laboratory values.Thus, the present invention encompasses intra-patient dose-escalation asdetermined by the skilled artisan. Determining appropriate dosages andregimens are well-known in the relevant art and would be understood tobe encompassed by the skilled artisan once provided the teachingsdisclosed herein.

Generally, for administration of the antibodies described herein(monospecific or bispecific), the candidate dosage can be administereddaily, every week, every other week, every three weeks, every fourweeks, every five weeks, every six weeks, every seven weeks, every eightweeks, every ten weeks, every twelve weeks, or more than every twelveweeks. For repeated administrations over several days or longer,depending on the condition, the treatment is sustained until a desiredsuppression of symptoms occurs or until sufficient therapeutic levelsare achieved, for example, to reduce symptoms associated with cancer.The progress of this therapy is easily monitored by conventionaltechniques and assays. The dosing regimen (including the anti-FLTmonospecific or bispecific antibody used) can vary over time.

In some embodiments, the candidate dosage is administered daily with thedosage ranging from about any of 1 μg/kg to 30 μg/kg to 300 μg/kg to 3mg/kg, to 30 mg/kg, to 100 mg/kg or more, depending on the factorsmentioned above. For example, daily dosage of about 0.01 mg/kg, about0.03 mg/kg, about 0.1 mg/kg, about 0.3 mg/kg, about 1 mg/kg, about 2.5mg/kg, about 3 mg/kg, about 5 mg/kg, about 10 mg/kg, about 15 mg/kg, andabout 25 mg/kg may be used.

In some embodiments, the candidate dosage is administered every weekwith the dosage ranging from about any of 1 μg/kg to 30 μg/kg to 300μg/kg to 3 mg/kg, to 30 mg/kg, to 100 mg/kg or more, depending on thefactors mentioned above. For example, a weekly dosage of about 0.01mg/kg, about 0.03 mg/kg, about 0.1 mg/kg, about 0.3 mg/kg, about 0.5mg/kg, about 1 mg/kg, about 2.5 mg/kg, about 3 mg/kg, about 5 mg/kg,about 10 mg/kg, about 15 mg/kg, about 25 mg/kg, and about 30 mg/kg maybe used.

In some embodiments, the candidate dosage is administered every twoweeks with the dosage ranging from about any of 1 μg/kg to 30 μg/kg to300 μg/kg to 3 mg/kg, to 30 mg/kg, to 100 mg/kg or more, depending onthe factors mentioned above. For example, a bi-weekly dosage of about0.1 mg/kg, about 0.3 mg/kg, about 1 mg/kg, about 2.5 mg/kg, about 3mg/kg, about 5 mg/kg, about 10 mg/kg, about 15 mg/kg, about 25 mg/kg,and about 30 mg/kg may be used.

In some embodiments, the candidate dosage is administered every threeweeks with the dosage ranging from about any of 1 μg/kg to 30 μg/kg to300 μg/kg to 3 mg/kg, to 30 mg/kg, to 100 mg/kg or more, depending onthe factors mentioned above. For example, a tri-weekly dosage of about0.1 mg/kg, about 0.3 mg/kg, about 1 mg/kg, about 2.5 mg/kg, about 3mg/kg, about 5 mg/kg, about 10 mg/kg, about 15 mg/kg, about 25 mg/kg,about 30 mg/kg, about 35 mg/kg, about 40 mg/kg, about 45 mg/kg, andabout 50 mg/kg may be used.

In some embodiments, the candidate dosage is administered every month orevery four weeks with the dosage ranging from about any of 1 μg/kg to 30μg/kg to 300 μg/kg to 3 mg/kg, to 30 mg/kg, to 100 mg/kg or more,depending on the factors mentioned above. For example, a monthly dosageof about 0.1 mg/kg, about 0.3 mg/kg, about 1 mg/kg, about 2.5 mg/kg,about 3 mg/kg, about 5 mg/kg, about 10 mg/kg, about 15 mg/kg, about 25mg/kg, about 30 mg/kg, about 35 mg/kg, about 40 mg/kg, about 45 mg/kg,and about 50 mg/kg may be used.

In other embodiments, the candidate dosage is administered daily withthe dosage ranging from about 0.01 mg to about 1200 mg or more,depending on the factors mentioned above. For example, daily dosage ofabout 0.01 mg, about 0.1 mg, about 1 mg, about 10 mg, about 50 mg, about100 mg, about 200 mg, about 300 mg, about 400 mg, about 500 mg, about600 mg, about 700 mg, about 800 mg, about 900 mg, about 1000 mg, about1100 mg, or about 1200 mg may be used.

In other embodiments, the candidate dosage is administered every weekwith the dosage ranging from about 0.01 mg to about 2000 mg or more,depending on the factors mentioned above. For example, weekly dosage ofabout 0.01 mg, about 0.1 mg, about 1 mg, about 10 mg, about 50 mg, about100 mg, about 200 mg, about 300 mg, about 400 mg, about 500 mg, about600 mg, about 700 mg, about 800 mg, about 900 mg, about 1000 mg, about1100 mg, about 1200 mg, about 1300 mg, about 1400 mg, about 1500 mg,about 1600 mg, about 1700 mg, about 1800 mg, about 1900 mg, or about2000 mg may be used.

In other embodiments, the candidate dosage is administered every twoweeks with the dosage ranging from about 0.01 mg to about 2000 mg ormore, depending on the factors mentioned above. For example, bi-weeklydosage of about 0.01 mg, about 0.1 mg, about 1 mg, about 10 mg, about 50mg, about 100 mg, about 200 mg, about 300 mg, about 400 mg, about 500mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, about 1000mg, about 1100 mg, about 1200 mg, about 1300 mg, about 1400 mg, about1500 mg, about 1600 mg, about 1700 mg, about 1800 mg, about 1900 mg, orabout 2000 mg may be used.

In other embodiments, the candidate dosage is administered every threeweeks with the dosage ranging from about 0.01 mg to about 2500 mg ormore, depending on the factors mentioned above. For example, tri-weeklydosage of about 0.01 mg, about 0.1 mg, about 1 mg, about 10 mg, about 50mg, about 100 mg, about 200 mg, about 300 mg, about 400 mg, about 500mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, about 1000mg, about 1100 mg, about 1200 mg, about 1300 mg, about 1400 mg, about1500 mg, about 1600 mg, about 1700 mg, about 1800 mg, about 1900 mg,about 2000 mg, about 2100 mg, about 2200 mg, about 2300 mg, about 2400mg, or about 2500 mg may be used.

In other embodiments, the candidate dosage is administered every fourweeks or month with the dosage ranging from about 0.01 mg to about 3000mg or more, depending on the factors mentioned above. For example,monthly dosage of about 0.01 mg, about 0.1 mg, about 1 mg, about 10 mg,about 50 mg, about 100 mg, about 200 mg, about 300 mg, about 400 mg,about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg,about 1000 mg, about 1100 mg, about 1200 mg, about 1300 mg, about 1400mg, about 1500 mg, about 1600 mg, about 1700 mg, about 1800 mg, about1900 mg, about 2000 mg, about 2100 mg, about 2200 mg, about 2300 mg,about 2400 mg, about 2500, about 2600 mg, about 2700 mg, about 2800 mg,about 2900 mg, or about 3000 mg may be used.

Kits

The invention also provides kits for use in the instant methods. Kits ofthe invention include one or more containers comprising the antibody(e.g., monospecific or bispecific) as described herein and instructionsfor use in accordance with any of the methods of the invention describedherein. Generally, these instructions comprise a description ofadministration of the antibody protein for the above describedtherapeutic treatments.

The instructions relating to the use of the antibody (e.g., monospecificor bispecific) as described herein generally include information as todosage, dosing schedule, and route of administration for the intendedtreatment. The containers may be unit doses, bulk packages (e.g.,multi-dose packages) or sub-unit doses. Instructions supplied in thekits of the invention are typically written instructions on a label orpackage insert (e.g., a paper sheet included in the kit), butmachine-readable instructions (e.g., instructions carried on a magneticor optical storage disk) are also acceptable.

The kits of this invention are in suitable packaging. Suitable packagingincludes, but is not limited to, vials, bottles, jars, flexiblepackaging (e.g., sealed Mylar or plastic bags), and the like. Alsocontemplated are packages for use in combination with a specific device,such as an inhaler, nasal administration device (e.g., an atomizer) oran infusion device such as a minipump. A kit may have a sterile accessport (for example the container may be an intravenous solution bag or avial having a stopper pierceable by a hypodermic injection needle). Thecontainer may also have a sterile access port (for example the containermay be an intravenous solution bag or a vial having a stopper pierceableby a hypodermic injection needle). At least one active agent in thecomposition is a bispecific antibody. The container may further comprisea second pharmaceutically active agent.

Kits may optionally provide additional components such as buffers andinterpretive information. Normally, the kit comprises a container and alabel or package insert(s) on or associated with the container.

The following examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.Indeed, various modifications of the invention in addition to thoseshown and described herein will become apparent to those skilled in theart from the foregoing description and fall within the scope of theappended claims.

EXAMPLES Example 1: Determination of Kinetics and Affinity of HumanCD70/CD70 Antibodies Interactions at 37° C.

The kinetics and affinity of anti-CD70 antibodies disclosed herein canbe measured on a Biacore T200 surface Plasmon resonance biosensor (GELifesciences, Piscataway N.J.).

Example 2: T-Cell Mediated Killing of RCC Cell Lines Using CD70-CD3Bispecific IgG In Vitro

Human anti-CD70 and human anti-CD3 (h2B4-VH-hnps VL-TK (“H2B4”))antibodies are expressed as human IgG2dA_D265A engineered with EEEE onone arm and RRRR on the other arm for bispecific exchange at positions223, 225, and 228 (e.g., (C223E or C223R), (E225E or E225R), and (P228Eor P228R)) in the hinge region and at position 409 or 368 (e.g., K409Ror L368E (EU numbering scheme)) in the CH3 region of human IgG2 (SEQ IDNO: 279). The CD70/CD3 bispecific antibody also has the mutation from Dto A at position 265 (EU numbering scheme).

CD3+ T cells from human PBMC are negatively selected using Pan T CellIsolation kit, human (Miltenyi, San Diego Calif.). Target expressing(786-0) cells and CD3+ T-cells are seeded on clear U-bottom plates at20000 and 100000 cells/well respectively. Cells are treated with 8-foldserially diluted bispecific antibody. RCC cell depletion is determinedby flow-cytometry analysis 24 hours after treatment. Cell depletion ismeasured by contrast to control treated cells. EC50 is calculated byPrism software.

Example 3: CD70-CD3 Bispecific IgG Induces Tumor Ablation in RCCSubcutaneous Xenograft Model

NOD scid gamma (NSG) mice are implanted with 786-0 tumors subcutaneouslyand once the tumors attained a volume of 200 mm³, the mice are dosedwith 20 million expanded T cells each intraperitoneally. Two days laterthe anti CD70 bispecific antibodies are dosed at 300, 100, or 30 ug/mLintravenously via tail vein injection to determine the optimalbispecific antibody dose.

Materials and Methods:

NOD scid gamma (NSG) mice are shaved and prepared for subcutaneous tumorimplant on the right flank. 786-0 tumor cells that are known to expressCD70 are expanded in RPMI supplemented with 10% FBS. On Day 0, 786-0cells are resuspended in serum-free RPMI at the required concentrationto inject 5 million cells per animal. Tumor cells are injected in 100 uLof serum-free RPMI combined with 100 uL Matrigel (Corning) per animalsubcutaneously. Day 0 baseline body weights are recorded for all animalsimmediately after tumor implant. Tumors are measured twice a weekstarting on Day 9 using Digimatic Calipers (Mitutoyo) and body weightsrecorded. On Day 14, when the tumors attained 200 mm³ (standard error8.39) 40 tumor-bearing mice are randomized to 4 groups of 10 mice each.T cells are thawed and expanded and then resuspended in serum-free RPMIat the required concentration to inject 20 million T cells per animal. Tcells are injected in 200 uL of serum-free RPMI per animalintraperitoneally. Two days later bispecifics are dosed intravenouslyvia tail vein at 300, 100, or 30 ug/mL per animal. Tumors are measuredand body weights recorded twice a week till the untreated group reachedthe study end-point (1500 mm³ tumor volume).

Tumor volumes (mean and error SEM) are plotted on GraphPad Prism andstatistics are calculated using one-way ANOVA with repeated measures.

Although the disclosed teachings have been described with reference tovarious applications, methods, kits, and compositions, it will beappreciated that various changes and modifications can be made withoutdeparting from the teachings herein and the claimed invention below. Theforegoing examples are provided to better illustrate the disclosedteachings and are not intended to limit the scope of the teachingspresented herein. While the present teachings have been described interms of these exemplary embodiments, the skilled artisan will readilyunderstand that numerous variations and modifications of these exemplaryembodiments are possible without undue experimentation. All suchvariations and modifications are within the scope of the currentteachings.

All references cited herein, including patents, patent applications,papers, text books, and the like, and the references cited therein, tothe extent that they are not already, are hereby incorporated byreference in their entirety. In the event that one or more of theincorporated literature and similar materials differs from orcontradicts this application, including but not limited to definedterms, term usage, described techniques, or the like, this applicationcontrols.

The foregoing description and Examples detail certain specificembodiments of the invention and describes the best mode contemplated bythe inventors. It will be appreciated, however, that no matter howdetailed the foregoing may appear in text, the invention may bepracticed in many ways and the invention should be construed inaccordance with the appended claims and any equivalents thereof.

It is claimed:
 1. An isolated antibody, which specifically binds toCluster of Differentiation 70 (CD70), wherein the antibody comprises aheavy chain variable (VH) region comprising a VH complementaritydetermining region one (CDR1), a VH CDR2, and a VH CDR3, and a lightchain variable (VL) region comprising a VL CDR1, a VL CDR2, and a VLCDR3, wherein: the VH CDR1 comprises the amino acid sequence of SEQ IDNO: 97, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 100,the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 102; or theVH CDR1 comprises the amino acid sequence of SEQ ID NO: 98, the VH CDR2comprises the amino acid sequence of SEQ ID NO: 101, the VH CDR3comprises the amino acid sequence of SEQ ID NO: 102; or the VH CDR1comprises the amino acid sequence of SEQ ID NO: 99, the VH CDR2comprises the amino acid sequence of SEQ ID NO: 100, the VH CDR3comprises the amino acid sequence of SEQ ID NO: 102; and wherein the VLCDR1 comprises the amino acid sequence of SEQ ID NO: 217, the VL CDR2comprises the amino acid sequence of SEQ ID NO: 218, and the VL CDR3comprises the amino acid sequence of SEQ ID NO:
 219. 2. The antibody ofclaim 1, wherein the VH region comprises the amino acid sequence of SEQID NO: 18 and the VL region comprises the amino acid sequence of SEQ IDNO:
 17. 3. A bispecific antibody wherein the bispecific antibody is afull-length antibody, comprising a first antigen binding site of thebispecific antibody specifically binding to a target antigen, andcomprising a second antigen binding site of the bispecific antibodycapable of recruiting the activity of a human immune effector cell byspecifically binding to an effector antigen located on the human immuneeffector cell, wherein the first antigen binding site comprises a heavychain variable (VH) region comprising a VH CDR1, VH CDR2, and VH CDR3and a light chain variable (VL) region comprising a VL CDR1, VL CDR2,and VL CDR3, wherein the VH region comprises the amino acid sequence ofSEQ ID NO: 18 and the VL region comprises the amino acid sequence of SEQID NO:
 17. 4. A bispecific antibody wherein the bispecific antibody is afull-length antibody, comprising a first antigen binding site of thebispecific antibody specifically binding to CD70, and comprising asecond antigen binding site of the bispecific antibody capable ofrecruiting the activity of a human immune effector cell by specificallybinding to an effector antigen located on the human immune effectorcell, wherein the first antigen binding site comprises a heavy chainvariable (VH) region comprising a VH complementarity determining regionone (CDR1), a VH CDR2, and a VH CDR3, and a light chain variable (VL)region comprising a VL CDR1, a VL CDR2, and a VL CDR3 wherein: the VHCDR1 comprises the amino acid sequence of SEQ ID NO: 97, the VH CDR2comprises the amino acid sequence of SEQ ID NO: 100, the VH CDR3comprises the amino acid sequence of SEQ ID NO: 102; or the VH CDR1comprises the amino acid sequence of SEQ ID NO: 98, the VH CDR2comprises the amino acid sequence of SEQ ID NO: 101, the VH CDR3comprises the amino acid sequence of SEQ ID NO: 102; or the VH CDR1comprises the amino acid sequence of SEQ ID NO: 99, the VH CDR2comprises the amino acid sequence of SEQ ID NO: 100, the VH CDR3comprises the amino acid sequence of SEQ ID NO: 102; and wherein the VLCDR1 comprises the amino acid sequence of SEQ ID NO: 217, the VL CDR2comprises the amino acid sequence of SEQ ID NO: 218, and the VL CDR3comprises the amino acid sequence of SEQ ID NO:
 219. 5. The bispecificantibody of claim 4, wherein the second antigen binding sitespecifically binds to the effector antigen CD3.
 6. The bispecificantibody of claim 5, wherein the second antigen binding site comprisesa) a heavy chain variable (VH) region comprising (i) a VH complementarydetermining region one (CDR1) comprising the sequence shown in SEQ IDNO: 267, 268, or 269; (ii) a VH CDR2 comprising the sequence shown inSEQ ID NO: 270 or 271; and (iii) a VH CDR3 comprising the sequence shownin SEQ ID NO: 272; and b) a light chain variable (VL) region comprising(i) a VL CDR1 comprising the sequence shown in SEQ ID NO: 273; (ii) a VLCDR2 comprising the sequence shown in SEQ ID NO: 274; and (iii) a VLCDR3 comprising the sequence shown in SEQ ID NO:
 275. 7. The bispecificantibody of claim 3, wherein both the first and the second antigenbinding sites of the bispecific antibody comprise amino acidmodifications at positions 223, 225, and 228 in the hinge region and atposition 409 or 368, according to the EU numbering scheme, in the CH3region of a human IgG2 comprising SEQ ID NO:
 279. 8. The bispecificantibody of claim 7, further comprising an amino acid modification atone or more of positions 265, 330 and 331 of the human IgG2.
 9. Anucleic acid encoding the antibody of claim
 1. 10. A vector comprisingthe nucleic acid of claim
 9. 11. A host cell comprising the nucleic acidof claim
 9. 12. A method of treating a subject in need thereofcomprising: a) providing the antibody of claim 1; and b) administeringsaid antibody to said subject.
 13. A pharmaceutical compositioncomprising the antibody of claim
 1. 14. A method of treating a conditionassociated with malignant cells expressing CD70 in a subject comprisingadministering to a subject in need thereof an effective amount of theantibody of claim
 1. 15. The method of claim 14, wherein the conditionis a cancer.
 16. The method of claim 15, wherein the cancer is an CD70related cancer selected from the group consisting of Renal CellCarcinoma, Glioblastoma, glioma such as low grade glioma, Non-Hodgkin'sLymphoma (NHL), Hodgkin's Disease (HD), Waldenstrom's macroglobulinemia,Acute Myeloid Leukemia, Multiple Myeloma, diffuse large-cell lymphoma,follicular lymphoma or Non-Small Cell Lung Cancer.
 17. A method ofinhibiting tumor growth or progression in a subject who has malignantcells expressing CD70, comprising administering to the subject in needthereof an effective amount of the pharmaceutical composition of claim13 to the subject.
 18. A method of inhibiting metastasis of malignantcells expressing CD70 in a subject, comprising administering to thesubject in need thereof an effective amount of the pharmaceuticalcomposition of claim 13 to the subject.
 19. A method of inducing tumorregression in a subject who has malignant cells expressing CD70,comprising administering to the subject in need thereof an effectiveamount of the pharmaceutical composition of claim 13 to the subject. 20.A method of producing an antibody, comprising culturing the host cell ofclaim 11 under conditions that result in production of the antibody, andisolating the antibody from the host cell or culture.
 21. The antibodyof claim 1, wherein: the VH CDR1 comprises the amino acid sequence ofSEQ ID NO: 97, the VH CDR2 comprises the amino acid sequence of SEQ IDNO: 100, and the VH CDR3 comprises the amino acid sequence of SEQ ID NO:102.
 22. An isolated antibody, which specifically binds to Cluster ofDifferentiation 70 (CD70), wherein the antibody comprises a heavy chainvariable (VH) region comprising a VH complementarity determining regionone (CDR1), a VH CDR2, and a VH CDR3, and a light chain variable (VL)region comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein the VLregion comprises the amino acid sequence of SEQ ID NO: 17 and the VHregion comprises the amino acid sequence of SEQ ID NO:
 18. 23. Thebispecific antibody of claim 4, wherein: the VH CDR1 comprises the aminoacid sequence of SEQ ID NO: 97, the VH CDR2 comprises the amino acidsequence of SEQ ID NO: 100, and the VH CDR3 comprises the amino acidsequence of SEQ ID NO:
 102. 24. A pharmaceutical composition comprisingthe antibody of claim
 2. 25. A pharmaceutical composition comprising theantibody of claim
 3. 26. A pharmaceutical composition comprising theantibody of claim
 4. 27. A pharmaceutical composition comprising theantibody of claim
 5. 28. A pharmaceutical composition comprising theantibody of claim
 6. 29. A pharmaceutical composition comprising theantibody of claim
 7. 30. A pharmaceutical composition comprising theantibody of claim
 8. 31. A pharmaceutical composition comprising theantibody of claim
 21. 32. A pharmaceutical composition comprising theantibody of claim
 22. 33. A pharmaceutical composition comprising theantibody of claim
 23. 34. A host cell comprising the vector of claim 10.35. A method of making the host cell of claim 11, comprising introducingthe nucleic acid of claim 9 into a host cell.
 36. A method of making thehost cell of claim 34, comprising introducing the vector of claim 11into a host cell.